Methods, devices, kits and compositions for detecting feline lungworm

Abstract

Compositions, devices, kits, and methods are provided for detecting the feline lungworm species Aelurostronglylus abstrusus ("A. abstrusus") in mammals. More particularly, polypeptides and polypeptide compositions, antibodies and antibody compositions, devices, kits, and methods are provided for detecting the presence or absence of A. abstrusus antibodies in a sample from a mammal, which may also contain one or more other feline lungworm, roundworm, hookworm, and whipworm antigens, diagnosing an infection with the lungworm A. abstrusus, and detecting, diagnosing, and treating an infection with the lungworm A. abstrusus. Compositions, vaccines, and methods including polypeptide or polynucleotide immunogens for treating and / or preventing feline lungworm infection in mammals are also provided.

Description

[Technical field]

[0001] cross reference This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 323,558, filed March 25, 2022, which is incorporated by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT A computer readable version of the Sequence Listing has been submitted with this application via electronic filing and is incorporated by reference in its entirety into this application. The Sequence Listing submitted with this application is contained in a text file entitled "20-1518-WO_Sequence-Listing.xml", created on March 21, 2023, and is 166,470 bytes in size.

[0003] Field of the Disclosure The present disclosure relates to compositions, devices, kits, and methods for detecting the feline lungworm species Aelurostrongylus abstrusus ("A. abstrusus") in a mammal, as well as compositions and vaccines comprising the compositions for treating and / or preventing feline lungworm infection in a mammal. More particularly, the disclosure relates to polypeptides and polypeptide compositions, antibodies and antibody compositions, devices, kits, and methods for detecting the presence or absence of A. abstrusus antibodies in a sample derived from a mammal, which may also contain one or more other feline lungworm, roundworm, hookworm, and whipworm antigens, as well as polypeptides and polynucleotides, and vaccines comprising the polypeptides or polynucleotides for treating and / or preventing feline lungworm infection in a mammal. [Background technology]

[0004] Feline pulmonary nematode disease is a veterinarily important parasitic lung disease. A. abstrusus is the best known feline lung nematode and is thought to be the most prevalent in domestic cats worldwide. Other feline lung nematodes include Oslerus rostratus ("O. rostratus"), Troglostrongylus brevior ("T. brevior"), Capillaria aerophila, and Paragonimus species. The nematode A. abstrusus has an indirect life cycle that involves cats as definitive hosts, terrestrial mollusks as intermediate hosts, and small vertebrates such as rodents, birds, lizards, and frogs as standby hosts. Adult worms are localized within the alveolar ducts and bronchioles of the feline host. Female worms hatch eggs within the lung parenchyma and small blood vessels, where first stage (L1) larvae develop. L1 larvae migrate through the bronchi and trachea to the pharynx where they are expectorated, swallowed, and released into the environment via feces. L1 larvae develop into infective L3 larvae that invade their intermediate hosts, which are then picked up by the host of entrainment. Ingestion of the host of entrainment by the feline host is the most well-recognized means of lung nematode transmission. When adult worms infest the lungs of the feline host, they can cause respiratory signs ranging from minor to interstitial bronchopneumonia, respiratory distress, and even death, especially in young, debilitated, or immunocompromised cats. Such a non-specific clinical pattern usually requires a high level of clinical awareness of the disease in order to initiate prompt treatment.

[0005] In addition to the lack of specific clinical signs, a major problem with the treatment and prevention of feline pulmonary strongyloidiasis is the absence of a clear diagnostic procedure. Current diagnostic methods for A. abstrusus infection mainly involve microscopic examination of fecal samples, either directly in fecal smears or after concentrating the eggs and parasites by flotation or sedimentation in a density medium. Despite the high adoption rate of this procedure, the methods have significant drawbacks. These microscopic methods are time-consuming and require specialized equipment. In addition, the accuracy of the results of these methods is highly dependent on the technique and expertise of the practitioner. For example, the Behrmann method is considered the standard method for A. abstrusus diagnosis and involves the recovery of larvae from feces. However, the Behrmann method can give false-negative results due to the low concentration of larvae in fecal samples. Furthermore, this technique requires specialized skills in distinguishing between A. abstrusus larvae and from other lungworm larvae, since A. abstrusus, O. rostrotus, and T. brevior share the same intermediate and parasite hosts and may cause mixed infections. It is not surprising that A. abstrusus infections are often undiagnosed or misdiagnosed with routine fecal testing. Summary of the Invention [Problem to be solved by the invention]

[0006] Fecal manipulation is unpleasant and dangerous. Sanitary and non-hazardous procedures for processing feces are often cumbersome and complicated. Such procedures may include weighing, centrifugation, and storage, and are difficult except in clinical laboratories equipped with appropriate equipment, protective facilities, and skilled technicians. Therefore, some reduction in the number of steps required to perform fecal testing and some reduction in contact between the tester and the test material is desirable. Clinical laboratories have used immunoassay methods to detect a variety of viruses, bacteria, and non-parasitic helminth parasites and organisms in feces. However, there remains a need for simple immunoassay methods for detecting A. abstrusus infection in feces, whole blood, or serum. In addition, there also remains a need for the treatment and / or prevention of feline lungworm infection, e.g., A. abstrusus infection, in mammals. [Means for solving the problem]

[0007] In one aspect, the disclosure includes a polypeptide comprising an epitope of an A. abstrusus antigen. In one embodiment, the polypeptide is selected from SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 truncation), SEQ ID NO:5 (T3 truncation), SEQ ID NO:6 (T4 truncation), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d40 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO: combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), 115 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) as listed herein, wherein (a) the amino acids W and F at positions 4 and 5, respectively, are retained, and at least one X at positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) the amino acids W and F at positions 4 and 5, respectively, are retained, and X at positions 1 and 3 is independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or a polypeptide comprising a sequence which is a conservative variant of one of these sequences;

[0008] In another aspect, the disclosure includes a combination polypeptide comprising one or more polypeptides, each polypeptide comprising an epitope of an A. abstrusus antigen. In one embodiment, the combination polypeptide comprises the amino acid sequences SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 truncation), SEQ ID NO:5 (T3 truncation), SEQ ID NO:6 (T4 truncation), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d21 peptide), SEQ ID NO:33 (d21 peptide), SEQ ID NO:34 (d21 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d15 peptide), SEQ ID NO: combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino-extended), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0 peptide), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), or one or more polypeptides comprising SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:115 (D8M9), SEQ ID NO:116 (D8M10), or XXXWF (SEQ ID NO:117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3;(c) the amino acids W and F at positions 4 and 5, respectively, are retained, and X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid. In another embodiment, the combination polypeptide comprises two or more polypeptides having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:78, or SEQ ID NO:79, or variants thereof differing only in conservative substitutions and / or modifications. The combination polypeptides include at least one epitope of an A. abstractus antigen having the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 78, and SEQ ID NO: 79, or variants thereof that differ only in conservative substitutions and / or modifications. Such combination polypeptides may be prepared by synthetic means or may be prepared using recombinant DNA technology;

[0009] In another aspect, the disclosure provides SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d15 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO:39 (d20 peptide), SEQ ID NO:40 (d37 peptide), SEQ ID combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:115 (D8M9), SEQ ID NO:116 ( D8M10), or a polypeptide comprising all or an antigenic portion of an amino acid sequence corresponding to one or more of the following: XXXWF (SEQ ID NO: 117) as listed herein, wherein: (a) the amino acids W and F at positions 4 and 5, respectively, are retained, and at least one X at positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) the amino acids W and F at positions 4 and 5, respectively, are retained, and X at positions 1 and 3 is independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or an antibody that specifically binds to a polypeptide comprising a sequence that is a conservative variant of one of these sequences. In a further embodiment, the antibody specifically binds to an antigen from a mammal infested with A. abstrusus, but does not specifically bind to an antigen from a mammal infected with hookworms, roundworms, whipworms, heartworms, and other feline lungworms;

[0010] In another aspect, the disclosure provides SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d15 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO:39 (d20 peptide), SEQ ID NO:40 (d37 peptide), SEQ ID combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino-extended), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), SEQ ID NO: 115 (D8M9), SEQ ID NO: No. 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in which: (a) the amino acids W and F at positions 4 and 5, respectively, are retained and at least one X at positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) the amino acids W and F at positions 4 and 5, respectively, are retained and X at positions 1 and 3 is independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or antibodies obtained by immunization with a polypeptide comprising a sequence which is a conservative variant of one of these sequences;

[0011] Within these related aspects, there are also provided DNA sequences encoding the above polypeptides, expression vectors containing these DNA sequences, and host cells transformed or transfected with such expression vectors.

[0012] In yet another aspect, the disclosure provides a device for detecting the presence or absence of an A. abstrusus antigen from a sample, comprising a solid support having thereon one or more of the following peptides: SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d7 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d4 peptide), SEQ ID NO: combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:11 a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4 or the amino acid F at position 5 is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or an antigenic portion thereof. The device may be, for example, but is not limited to, an ELISA device, such as a lateral flow immunoassay device or a microtiter plate device. Mammalian samples that may be interrogated for A. abstrusus by the device include, but are not limited to, blood and associated fluids such as serum and plasma, fluids and tissues obtained from throat swabs, transtracheal aspirates, endotracheal swabs, bronchoalveolar lavage, pleural effusions, and other materials obtained from all tissues, such as, for example, airways, feces, and tissues from the small intestine, large intestine, cecum, colon, rectum, or other tissues obtained from the digestive tract. The device may, but does not necessarily, further comprise one or more reagents for detection of one or more of the group consisting of one or more non-A. abstrusus helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, and one or more bacteria;

[0013] In yet another aspect, the present disclosure provides a method for detecting the presence or absence of A. abstrusus in a sample. The sample may be obtained from a mammal, such as a dog, a cat, a pig, a cow, or a human. In one aspect, the method is performed to test a serum sample for A. abstrusus antigens. However, the method is not limited to being performed to test a serum sample. Thus, in addition to serum, the sample may be, but is not limited to, blood and related fluids, such as plasma, fluids and tissues obtained from throat swabs, transtracheal aspirates, endotracheal swabs, bronchoalveolar lavage, pleural effusions, and other materials obtained from all tissues, such as tissues from the airways, small intestine, large intestine, cecum, colon, rectum, or other tissues obtained from the digestive tract, for example. In one embodiment, the steps of the method include contacting the sample with one or more of the polypeptides of the present disclosure; forming an antibody-polypeptide complex, if present, in the presence of an antibody in the sample; and detecting the presence or absence of the antibody-polypeptide complex, if present. In another embodiment, the method steps include contacting the sample with one or more of the disclosed combination polypeptides; forming an antibody-combination polypeptide complex in the presence of an antibody in the sample, if present; and detecting the presence or absence of an antibody-combination polypeptide complex, if present. In another embodiment, the method steps include contacting the sample with one or more of the disclosed antibodies; forming an antibody-polypeptide complex in the presence of an A. abstrusus antigen in the sample, if present; and detecting the presence or absence of an antibody-polypeptide complex, if present.The method may further include one or more of the optional steps of diagnosing whether the mammal has or does not have an A. abstrusus infection, the optional step of treating a mammal diagnosed as having an A. abstrusus infection, and determining whether nucleic acid derived from A. abstrusus is present in the same sample contacted with the polypeptide, or in some other sample derived from the mammal, for the purpose of detecting the presence or absence of A. abstrusus.

[0014] The method can also be used to check for environmental contamination by conservative variants of one of these sequences.The environmental samples that can be checked by the device include, but are not limited to, soil, decomposition material, or fecal or body fluid material from residential environments, including farms, gardens, sandboxes, and playgrounds.Testing locations can also include parks, beaches, forests, farms, or other locations that are exposed to feces or other body material from dogs, cats, or other intermediate and standby hosts of A.abstrusus.Feces from indoor and outdoor trash cans can also be checked.

[0015] In yet another aspect, the present disclosure includes a kit for carrying out one or more steps of the method of the present disclosure. The kit can optionally include, for example, one or more of the device and composition of the present disclosure, and instructions for carrying out the method of the present disclosure. The kit can optionally further include, for example, one or more indicator reagents, one or more antibody-labeled compounds, one or more antibodies, one or more antigen capture reagents, one or more inhibitors, and one or more wash reagents that are used as part of the device and / or used in carrying out the method.

[0016] In a further aspect, the present disclosure provides pharmaceutical compositions comprising one or more polypeptides containing one or more epitopes of A. abstrusus antigens, or polynucleotides encoding one or more of these polypeptides, and a physiologically acceptable carrier, as well as vaccines comprising one or more of the above polypeptides and an adjuvant for enhancing the immune response.

[0017] These and other aspects of the present disclosure will become apparent upon reference to the following detailed description and accompanying drawings. All references disclosed herein are incorporated by reference in their entirety as if each was individually incorporated. [Brief description of the drawings]

[0018] [Figure 1A] (A) SEQ ID NO:1, the nucleotide sequence of the cDNA deduced from A. abstrusus, and SEQ ID NO:124, the encoded amino acid sequence. [Figure 1B-1] (B) SEQ ID NO: 20, a construct for expressing rTDX1557 (the amino acid sequence of rTDX1557 is SEQ ID NO: 3). [Figure 1B-2] Continued from Figure 1B-1 [Figure 1B-3] Continued from Figure 1B-2 [Figure 1B-4] Continued from Figure 1B-3 [Figure 1B-5] Continued from Figure 1B-4 [Figure 1C] (C) SDS-PAGE gel analysis of supernatants from mammalian expression system; and anti-HIS immunoblot analysis of (lane 1) prestained marker; (lane 2) recombinant TDX1557 (10ug / mL); (lane 3) recombinant TDX1557 (5ug / mL); and (lane 4) recombinant TDX1557 (10ug / mL). Lanes were probed with alkaline phosphatase tagged mAb HIS followed by development with BCIP / NBT. [Diagram 2]Part (A) shows a comparison of ELISA assays with A. abstrusus lysate and TDX1557 (2M2) with larval shedding (Behrman method) in a high shedding cat (BigMac). Part (B) shows a comparison of ELISA assays with A. abstrusus lysate and TDX1557 (2M2) with larval shedding (Behrman method) in a low shedding cat (Burrito). The X-axis indicates days post infection (DPI). The Y1 and Y2 axes represent OD values ​​and shedding rates, respectively. Cats were infected with L3 larvae and fecal samples and blood were collected weekly after infection. Cats were treated with antiparasitic drugs up to day 90. Larval shedding was performed using the standard Behrman method. ELISA assays with serum samples were performed with larval lysate and rTDX1557. Results indicate that although larval excretion was inconsistent, lysate and rTDX1557 ELISA assays showed a gradual increase in antibody titers to the worms, and a drop in antibody titers after day 90, indicating that the assays were responsive to treatment. [Diagram 3] Figure 1 shows that anti-TDX1557 (2M2) was not found to cross-react with D. immitis antigens. Part A shows SDS-PAGE analysis of A. abstrusus lysate and D. immitis lysate. Part B shows immunoblot analysis of A. abstrusus lysate and D. immitis lysate probed with rabbit pAB against TDX1557 (anti-TDX1557). Rabbit pAB against TDX1557 recognized a single band at approximately 62 kDa in A. abstrusus lysate but not in D. immitis lysate. The positive control is recombinant TDX1557, which also migrates to approximately 62 kDa. [Figure 4]1 shows that anti-TDX1557(2M2) was not found to cross-react with Ascaris antigens using immunoblot analysis of A.abstrusus lysates and Ascaris lysates probed with rabbit pAB against TDX1557 (anti-TDX1557). Rabbit pAB against TDX1557 recognized a single band at about 62 kDa in A.abstrusus lysates but not in Ascaris lysates. The positive control is recombinant TDX1557, which also migrates to about 62 kDa. [Figure 5A-C] Part (A) shows a comparison of BigMac, a high shedding species, with fecal samples versus ELISA with rTDX1557 and A.abstrusus lysate from cat serum samples. Inconsistencies in shedding rates were observed with fecal samples, whereas antibody responses to rTDX1557 or A.abstrusus lysate show a stable evolution. The antibody response drops with antiparasitic drugs after day 90, indicating that antibodies against rTDX1557 or lysate respond to treatment. Part B shows in tabular form the indirect ELISA for rTDX1557 using serum samples, larval shedding in fecal samples, and SNAP® results with serum samples from BigMac collected after different time periods. Part C shows photographic results for serum samples collected from a high shedding species, Big Mac, over a period of time (days 0, 7, 14, 28, 63, 91, 98, 126, 168, and 182) and subjected to SNAP analysis. [Figure 5D] Part D shows the SNAP results for serum samples from non-infected cat field samples that were also indirect ELISA negative for rTDX 1557. Nine of ten of these samples were found to be negative. [Figure 6] 1 shows the amino acid sequences for polypeptide rTDX1557 (SEQ ID NO:3), and truncated polypeptides T2 (SEQ ID NO:4), T3 (SEQ ID NO:5), and T4 (SEQ ID NO:6). The sequences include an IgG-like signal sequence, a HIS tag, and an ADX18 epitope tag. [Figure 7] FIG. 1 shows the predicted secondary structures for the polypeptide rTDX1557 and the truncated polypeptides T2, T3, and T4, as well as SDS-PAGE analysis of the expression of each truncated peptide. [Figure 8] Figure 1. Assessment of truncated forms of 2M2 (TDX1557) using indirect ELISA assay. Recombinant polypeptide 2M2 (TDX1557) and truncated polypeptides T2, T3, and T4 were assessed in indirect ELISA using rabbit pAb against TDX1557, timed cat serum samples (Burrito DPI 98, French Fry DPI 91), and negative cat serum samples (specific pathogen free (SPF) 16CMF3, SPF 16CSL3). Serially diluted serum samples were tested at different concentrations (0.5, 1.5, and 3ug / ml) of recombinant truncated forms. Rabbit pAb against TDX1557 reacted with all four polypeptides. However, experimental infection samples did not show any reactivity with truncated polypeptide T3, indicating that prominent epitopes are present only at the carboxy terminus of the protein. The SPF negative controls (SPF 16CMF3 and SPF 16CSL3) show no reactivity to any of the antigens. An increasing rate of titration was also observed based on antigen concentration and dilution. [Figure 9]Figure 1 shows the assessment of truncated forms of 2M2 (TDX1557) using an indirect ELISA assay. Recombinant 2M2 and truncated peptides T2, T3, and T4 were assessed in an indirect ELISA assay using rabbit pAb, time-course samples (Burrito DPI 98, French Fry DPI 91), and negative samples (SPF 16CMF3, SPF 16CSL3).Serially diluted serum samples were incubated with different concentrations (0.5, 1.5, and 3ug / ml) of the recombinant truncated forms (A01 is SEQ ID NO:27; A02 is SEQ ID NO:28; A03 is SEQ ID NO:29; A04 is SEQ ID NO:30; A05 is SEQ ID NO:31; A06 is SEQ ID NO:32; A07 is SEQ ID NO:33; A08 is SEQ ID NO:34; A09 is SEQ ID NO:35; A10 is SEQ ID NO:36; A11 is SEQ ID NO:37; A12 is SEQ ID NO:38; B01 is SEQ ID NO:39 ;B02 is SEQ ID NO:40;B03 is SEQ ID NO:41;B04 is SEQ ID NO:42;B05 is SEQ ID NO:43;B06 is SEQ ID NO:44;B07 is SEQ ID NO:45;B08 is SEQ ID NO:46;B09 is SEQ ID NO:47;B10 is SEQ ID NO:48;B11 is SEQ ID NO:49;B12 is SEQ ID NO:50;C01 is SEQ ID NO:51;C02 is SEQ ID NO:52;C03 is SEQ ID NO:53;C04 is SEQ ID NO:54;C05 is SEQ ID NO:55;C06 is SEQ ID NO: No. 56;C07 is SEQ ID NO:57;C08 is SEQ ID NO:58;C09 is SEQ ID NO:59;C10 is SEQ ID NO:7;C11 is SEQ ID NO:8;C12 is SEQ ID NO:9;D01 is SEQ ID NO:10;D02 is SEQ ID NO:11;D03 is SEQ ID NO:12;D04 is SEQ ID NO:13;D05 is SEQ ID NO:14;D06 is SEQ ID NO:15;D07 is SEQ ID NO:16;D08 is SEQ ID NO:17;D09 is SEQ ID NO:60;D10 is SEQ ID NO:61;D D11 is SEQ ID NO:62; D12 is SEQ ID NO:63; E01 is SEQ ID NO:64; E02 is SEQ ID NO:65; E03 is SEQ ID NO:66; E04 is SEQ ID NO:67; E05 is SEQ ID NO:68; E06 is SEQ ID NO:69; E07 is SEQ ID NO:70; E08 is SEQ ID NO:71; E09 is SEQ ID NO:72; E10 is SEQ ID NO:73; E11 is SEQ ID NO:74; E12 is SEQ ID NO:75; F01 is SEQ ID NO:76; and F02 is SEQ ID NO:27.) Rabbit pAbs against TDX1557 reacted with 2M2 and all four different truncations.However, none of the experimentally infected samples showed reactivity with the truncated T3, indicating that the prominent epitope is present only at the carboxy terminus of the protein. The SPF negative controls (SPF 16CMF3 and SPF 16CSL3) show no reactivity to any of the antigens. An increasing rate of titration was also observed based on antigen concentration and dilution. [Figure 10]1 shows the results of peptide array assessment of TDX1557 ELISA-positive naturally infected cat serum samples (8 cases) showing significant reactivity to peptides D1 to D8 and C10 to C12. The amino acid sequences of representative reactive peptides D2 (SEQ ID NO: 11), D3 (SEQ ID NO: 12), D4 (SEQ ID NO: 13); D6 (SEQ ID NO: 15), D7 (SEQ ID NO: 16), D8 (SEQ ID NO: 17), C10 (SEQ ID NO: 7), C11 (SEQ ID NO: 8), and C12 (SEQ ID NO: 9) are shown.(A01 is SEQ ID NO:27; A02 is SEQ ID NO:28; A03 is SEQ ID NO:29; A04 is SEQ ID NO:30; A05 is SEQ ID NO:31; A06 is SEQ ID NO:32; A07 is SEQ ID NO:33; A08 is SEQ ID NO:34; A09 is SEQ ID NO:35; A10 is SEQ ID NO:36; A11 is SEQ ID NO:37; A12 is SEQ ID NO:38; B01 is SEQ ID NO:39; B02 is SEQ ID NO:40; B03 is SEQ ID NO:41; B04 is SEQ ID NO: No. 42;B05 is SEQ ID NO:43;B06 is SEQ ID NO:44;B07 is SEQ ID NO:45;B08 is SEQ ID NO:46;B09 is SEQ ID NO:47;B10 is SEQ ID NO:48;B11 is SEQ ID NO:49;B12 is SEQ ID NO:50;C01 is SEQ ID NO:51;C02 is SEQ ID NO:52;C03 is SEQ ID NO:53;C04 is SEQ ID NO:54;C05 is SEQ ID NO:55;C06 is SEQ ID NO:56;C07 is SEQ ID NO:57; C08 is SEQ ID NO:58; C09 is SEQ ID NO:59; C10 is SEQ ID NO:7; C11 is SEQ ID NO:8; C12 is SEQ ID NO:9; D01 is SEQ ID NO:10; D02 is SEQ ID NO:11; D03 is SEQ ID NO:12; D04 is SEQ ID NO:13; D05 is SEQ ID NO:14; D06 is SEQ ID NO:15; D07 is SEQ ID NO:16; D08 is SEQ ID NO:17; D09 is SEQ ID NO:60; D10 is SEQ ID NO:61; D11 is SEQ ID NO:62 D12 is SEQ ID NO:63; E01 is SEQ ID NO:64; E02 is SEQ ID NO:65; E03 is SEQ ID NO:66; E04 is SEQ ID NO:67; E05 is SEQ ID NO:68; E06 is SEQ ID NO:69; E07 is SEQ ID NO:70; E08 is SEQ ID NO:71; E09 is SEQ ID NO:72; E10 is SEQ ID NO:73; E11 is SEQ ID NO:74; E12 is SEQ ID NO:75; F01 is SEQ ID NO:76; and F02 is SEQ ID NO:27. [Figure 11]Figure 1 shows the assessment of peptide arrays with experimentally infected cat serum samples showing positive ELISA assay results with rTDX1557. The serum samples show significant reactivity to peptides. The amino acid sequences of portions of representative reactive peptides D2 (SEQ ID NO: 11), D3 (SEQ ID NO: 12), D4 (SEQ ID NO: 13); D6 (SEQ ID NO: 15), D7 (SEQ ID NO: 16), D8 (SEQ ID NO: 17), C10 (SEQ ID NO: 7), C11 (SEQ ID NO: 8), C12 (SEQ ID NO: 9) are shown.(A01 is SEQ ID NO:27; A02 is SEQ ID NO:28; A03 is SEQ ID NO:29; A04 is SEQ ID NO:30; A05 is SEQ ID NO:31; A06 is SEQ ID NO:32; A07 is SEQ ID NO:33; A08 is SEQ ID NO:34; A09 is SEQ ID NO:35; A10 is SEQ ID NO:36; A11 is SEQ ID NO:37; A12 is SEQ ID NO:38; B01 is SEQ ID NO:39; B02 is SEQ ID NO:40; B03 is SEQ ID NO:41; B04 is SEQ ID NO: No. 42;B05 is SEQ ID NO:43;B06 is SEQ ID NO:44;B07 is SEQ ID NO:45;B08 is SEQ ID NO:46;B09 is SEQ ID NO:47;B10 is SEQ ID NO:48;B11 is SEQ ID NO:49;B12 is SEQ ID NO:50;C01 is SEQ ID NO:51;C02 is SEQ ID NO:52;C03 is SEQ ID NO:53;C04 is SEQ ID NO:54;C05 is SEQ ID NO:55;C06 is SEQ ID NO:56;C07 is SEQ ID NO:57; C08 is SEQ ID NO:58; C09 is SEQ ID NO:59; C10 is SEQ ID NO:7; C11 is SEQ ID NO:8; C12 is SEQ ID NO:9; D01 is SEQ ID NO:10; D02 is SEQ ID NO:11; D03 is SEQ ID NO:12; D04 is SEQ ID NO:13; D05 is SEQ ID NO:14; D06 is SEQ ID NO:15; D07 is SEQ ID NO:16; D08 is SEQ ID NO:17; D09 is SEQ ID NO:60; D10 is SEQ ID NO:61; D11 is SEQ ID NO:62 D12 is SEQ ID NO:63; E01 is SEQ ID NO:64; E02 is SEQ ID NO:65; E03 is SEQ ID NO:66; E04 is SEQ ID NO:67; E05 is SEQ ID NO:68; E06 is SEQ ID NO:69; E07 is SEQ ID NO:70; E08 is SEQ ID NO:71; E09 is SEQ ID NO:72; E10 is SEQ ID NO:73; E11 is SEQ ID NO:74; E12 is SEQ ID NO:75; F01 is SEQ ID NO:76; and F02 is SEQ ID NO:27. [Figure 12]Figure 1 shows a comparison of the immunogenicity of peptides D6, D7, and D8 (D6: KNLTEMATRASKSWF is SEQ ID NO: 15; D7: MATRASKSWFDELKK is SEQ ID NO: 16; D8: SKSWFDELKKFGVPP is SEQ ID NO: 17), A. abstrusus lysate, and rTDX1557 through ELISA assay using rabbit pAB against rTDX1557. Peptides D6, D7, and D8 detected rabbit pAB against rTDX1557 early, indicating their diagnostic utility for infection. ELISA assay with A. abstrusus larvae lysate and rTDX1557 shows a gradual increase in antibody titers. Antibody titers drop at 90 days after treatment with antiparasitic drugs. [Figure 13] 1 shows the predicted secondary structure for polypeptide 2M2 (SEQ ID NO: 124) using Protter software. Based on the predicted structure, polypeptide 2M2 is a secreted protein. Shows the alignment of the amino acid sequences of immunogenic peptides D6, D7, and D8 (D6 is SEQ ID NO: 15; D7 is SEQ ID NO: 16; D8 is SEQ ID NO: 17) compared to peptide D678 (CPAQKNLTEMATRASKSWFDELKKFG is SEQ ID NO: 18), with overlapping sequences underlined. The portion of peptide sequence 2M2 that corresponds to polypeptide D678 is circled. [Figure 14]Figure 1 shows the immunogenicity comparison of polypeptide D678, A.abstrusus lysate and rTDX1557 through ELISA assay using rabbit pAB against rTDX1557.Plates were coated with polypeptide D678, A.abstrusus lysate and rTDX1557 and washed.Then, cat serum from cat Big Mac, Burrito, Chicken Nugget, French Fry, Taco and Tortilla was applied to the plate.Plates were washed and then rabbit pAB was applied to the plate.Secondary antibody was used for detection.Peptide D678 showed similar reactivity with A.abstrusus lysate. [Figure 15] FIG. 1 shows a schematic representation of extended D678 synthetic peptide sequences for 2M2, 2M2 amino 2Cys, D678 (SEQ ID NO: 78), D678 carboxyl (SEQ ID NO: 80), and D678 amino (SEQ ID NO: 79). [Figure 16A] FIG. 1 shows the results of an assessment of the immunogenicity of D678 extended peptides, D678 carboxyl and D678 amino, via an indirect ELISA assay using rabbit pAb against rTDX1557 and serum samples from time-course and SPF cats. [Figure 16B] FIG. 1 shows schematic representations of the truncated polypeptides of 2M2, 2M2 Amino 2Cys, CpepC, T4 WO IDR, T4 C2S, T4 1A, T4 1B, and T4 1C, and the extended peptides of D678, D678 carboxyl and D678 amino. [Figure 16C1]FIG. 2 shows the construction of SEQ ID NO:2 and epitopes 2M2 full length (TDX1557), 2M2 T2 (TDX1637), 2M2 T3 (TDX1674), 2M2 T4 (TDX1675), 2M2 amino 2Cys, 2M2 CpepC, and 2M2 Wo, as well as four Cys-Ser mutants of 2M2 T4. The four Cys-Ser mutants are 2M2 T4 WO IDR, 2M2 T4 1A, 2M2 T4 1B, and 2M2 T4 1C. [Figure 16C2] FIG. 1 shows the amino acid sequences for Cys-Ser mutants 2M2 T4 WO IDR (2M2_Amino2Cys_pCDNA34 is SEQ ID NO: 118; 2M2_CpepC_pcDNA34 is SEQ ID NO: 119; 2M2_T4C2S is SEQ ID NO: 120), 2M2 T4 1A (SEQ ID NO: 121), 2M2 T4 1B (SEQ ID NO: 122), and 2M2 T4 1C (SEQ ID NO: 123). [Figure 16D] Figure 1 shows the results of an assessment of the immunogenicity of the truncations 2M2 amino2Cys and 2M2 CpepC (2M2_Amino2Cys_pcDNA34 is SEQ ID NO: 118; 2M2_CpepC_pcDNA34 is SEQ ID NO: 119) via an indirect ELISA assay using rabbit pAbs against rTDX1557 and serum samples from aged and SPF cats. No reactivity was observed with samples from aged cats. [Figure 16E] Figure 1 shows the results of an assessment of the immunogenicity of the truncated form 2M2 T4 C2S (2M2_T4C2S is SEQ ID NO: 120) via an indirect ELISA assay using rabbit pAb against rTDX1557 and serum samples from aged and SPF cats. No reactivity was observed with samples from aged cats. [Figure 17]Immunoblot analysis of A. abstrusus lysates, rTDX1557 (positive control), and fecal samples from Big Mac, a high larval shedding cat, probed with rabbit pAB against rTDX1557 (anti-TDX1557) at days 36, 64, 141, and 180. Rabbit pAB against TDX1557 recognized a single band of approximately 62 kDa in A. abstrusus lysates, rTDX1557, and BigMac fecal samples at days 36, 64, and 141. BigMac was treated with antiparasitic drugs 90 days after infection. [Figure 18] The strong reduction in OD values ​​by D8M7 (tryptophan to alanine) and D8M8 (phenylalanine to alanine) compared to D8M0 indicates that the amino acids at positions 7 and 8 are crucial for binding of antibodies in serum from cats infected with A. abstrusus. The increase in OD values ​​by D8M10 compared to D8M0 indicates that the substitution of glutamic acid (a negatively charged amino acid) with alanine (a small non-polar amino acid) enhanced the binding of the peptide to antibodies in serum from cats infected with A. abstrusus. Similar results were obtained when the peptide was examined with serum from cats experimentally infected with A. abstrusus at various time points (data not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] introduction The present disclosure is generally directed to methods, devices, kits, and compositions for detecting A. abstrusus in a sample, such as serum, obtained from a mammal. In particular, the disclosure relates to A. abstrusus polypeptides and conservative variants thereof, polynucleotides encoding these polypeptides, and oligonucleotides that specifically bind to these polynucleotides, antibodies raised against and that specifically bind to these polypeptides, and methods, devices, and kits for detecting A. abstrusus.

[0020] The present disclosure offers a superior alternative to these existing microscopic screening methods. This is true because the present disclosure offers compositions, devices, kits, and methods for detecting the presence or absence of A. abstrusus in samples from mammals that (1) are easy to use and provide consistent and reliable results; (2) allow for confirmation of the absence or presence of A. abstrusus in mammals, regardless of whether the mammal is infected with hookworms, roundworms, whipworms, heartworms, and / or other lungworms; and (3) can detect A. abstrusus prior to the time that A. abstrusus larvae first appear in the feces of an infected host.

[0021] The present disclosure is based in part on the discovery of the unexpected properties of the composition of the present disclosure.Specifically, the present disclosure has determined that the antibody of the present disclosure raised against the polypeptide of the present disclosure can be used to capture and detect A.abstrusus antigen in a mammal, even when the mammal is also infested with one or more of hookworm, roundworm, whipworm, heartworm, and / or other lungworm of cats.This specificity to A.abstrusus is surprising, because whipworm, roundworm, hookworm, heartworm, and other lungworm of cats are all related nematodes, and the antibody raised against the protein isolated from any one of these worms is expected to cross-react with one or more of other worms, host antigens, or other host components.

[0022] It was further determined that the antibodies could be used to capture and detect A. abstrusus antigens in mammals as early as 14 days after the mammal was first infected with A. abstrusus. This ability to detect A. abstrusus so early after infection and prior to the appearance of any A. abstrusus larvae in the feces of infected mammals is surprising, since A. abstrusus larvae do not generally appear in the feces of an infected host until about 5-6 weeks after the host is infected.

[0023] Thus, the present disclosure includes methods, devices, compositions, and kits that use antibodies and / or fragments thereof to specifically capture and detect A.abstrusus antigens in mammals that may also be infested with one or more of roundworms, hookworms, whipworms, heartworms, and / or other lungworms. The ability of the present disclosure to detect and diagnose A.abstrusus even when one or more other types of helminths are also present allows the mammalian caregiver the opportunity to optimally select a treatment to remove A.abstrusus from the mammal. Furthermore, the ability of the present disclosure to detect A.abstrusus in some cases as early as 14 days after the mammal is first infected allows the caregiver the possibility of initiating such treatment before A.abstrusus makes the mammal seriously ill. Intervention before the appearance of larvae in the feces will also greatly reduce or eliminate the possibility of the infestation spreading to other animals.

[0024] Definitions and Use of Terms The term "composition of the disclosure" refers to all of the nucleic acids, polypeptides, antibodies, and mixtures containing one or more of the nucleic acids, polypeptides, and antibodies, and one or more other compounds, that can be used to detect the presence or absence of A. abstrusus in a sample obtained from a mammal by practicing the methods of the disclosure, as expressly described, implicitly included, or otherwise disclosed herein.

[0025] A "sample from a mammal" in which A. abstrusus may be detected according to the present disclosure includes all body components, such as any fluid, solid, cell, or tissue, and extracts thereof, that may contain A. abstrusus antigens. Thus, exemplary samples include, but are not limited to, blood and blood components such as plasma, fluids and tissues obtained from throat swabs, transtracheal aspirates, endotracheal swabs, bronchoalveolar lavage, pleural effusions, and serum and whole tissues, such as tissues from the airways, small intestine, large intestine, cecum, colon, rectum, or other materials obtained from other tissues, for example, from the digestive tract. The sample may be taken directly from the mammal, or the sample may be taken from anything that has come into contact with the mammal. For example, the sample may be feces that has just been excreted from the mammal, or feces that has been excreted from the mammal and is decaying. As another example, a sample may include soil, sludge, sediment, plant material, or any other material that may be left behind by a mammal and that may be mixed with body components, such as feces. Whatever the origin or contents of the sample, the sample is sometimes referred to herein as a "mammalian sample," "test sample," or "subject sample."

[0026] As used herein, "nucleic acid" is synonymous with, and is therefore used interchangeably with, "gene," "DNA," "cDNA," "EST," "polynucleotide," "oligonucleotide," "polynucleic acid," "RNA," and "mRNA." Nucleic acids can be in double-stranded or single-stranded form. Furthermore, nucleic acids can be either naturally isolated, such as from whole A.abstrusus or a portion thereof, or artificially synthesized, for example, in a recombinant host organism, such as by utilizing PCR-based techniques, by creating a transgenic organism that synthesizes the nucleic acid, by using a DNA synthesizer, or by any other molecular-based technique, or by any other artificial means known to those of skill in the art.

[0027] "Polypeptide", "peptide" and "protein" are synonymous terms used interchangeably herein to refer to a polymer of amino acid residues. The polypeptides, peptides and proteins of the present disclosure may be either naturally isolated, such as from whole A.abstrusus or from a portion of A.abstrusus, or artificially synthesized, either in a recombinant host organism or by any other artificial means known to those skilled in the art. A polypeptide that includes an epitope may consist entirely of the epitope or may contain additional sequences. The additional sequences may be derived from a natural antigen or from a heterologous antigen, and such sequences may (but need not) be antigenic.

[0028] The term "antibody" or "antibody of the present disclosure" refers to any antibody capable of specifically binding to one or more A.abstrusus antigens, but not to any antigens derived from hookworms, roundworms, whipworms, or heartworms. The antibodies of the present disclosure can be raised against one or more immunogenic polypeptides of the present disclosure. It is understood that, unless otherwise stated, the antibodies of the present disclosure can include a mixture of two or more different types of antibodies. For example, an antibody can be a mixture of two types of antibodies, where one of the two types specifically binds to a particular antigen and the other of the two types specifically binds to some other antigen.

[0029] An "immunogenic polypeptide of the disclosure," and more simply, a "polypeptide of the disclosure," is an immunogen against which an antibody of the disclosure can be raised and / or which specifically binds to A. abstrusus antibodies present in a sample. All "polypeptides of the disclosure" are immunogenic and therefore can be used to elicit an immune response in a host animal that produces an antibody of the disclosure. Unless otherwise stated, it is understood that a polypeptide of the disclosure can be a component of a mixed composition of multiple components.

[0030] An "immunogen" is any agent, such as an immunogenic polypeptide of the disclosure, that is capable of eliciting an immune response in an animal exposed to the agent.

[0031] As used herein, the term "A. abstrusus" refers to nematodes such as lung worms of the order Rhabditida. Thus, exemplary lung worms include Dictyocaulus viviparus, Angiostrongylus cantonensis, and Angiostrongylus vasorum. Furthermore, as used herein, the term "lung worms" does not refer to the entire phylum Nematoda. For example, "lung worms" does not include any members of the genera Ancylostoma, Uncinaria, Necator, Toxocara, Toxascaris, Ascaris, or Dirofilaria.

[0032] A "pulmonary worm antigen" or "pulmonary worm antigen" is any A. abstrusus product that is present in serum, blood, or material from the respiratory tract or feces of a mammal having a lungworm infection and that can be specifically bound by one or more of the antibodies of the present disclosure. For example, a lungworm antigen can be, but is not limited to, one or more of the polypeptides of the present disclosure.

[0033] A "lungworm antibody" or "antibody to lungworm" is any lungworm antibody that is present in the serum or blood of a mammal with an A. abstrusus infection and to which one or more of the polypeptides of the present disclosure can specifically bind. For example, a lungworm antibody can be, but is not limited to, an antibody that binds to one or more of the polypeptides of the present disclosure.

[0034] "Specific to", "specifically binds" and "stably binds" mean that a particular composition of the present disclosure, such as an antibody, polypeptide or oligonucleotide of the present disclosure, recognizes and binds, for example, one or more other agents with greater affinity than at least one other agent. By way of example, an antibody of the present disclosure is said to be "specific to", "specifically binds" and "stably binds" a lungworm antigen whenever it is capable of recognizing and binding to these lungworm antigens with greater affinity than any other antigens derived from a parasitic helminth other than lungworm. Such binding specificity can be examined using methods well known in the art, such as ELISA or radioimmunoassay (RIA). Based on the information observed regarding the binding specificity of a particular composition of the present disclosure, the method of the present disclosure can be carried out under conditions that allow the composition to bind to one or more particular agents (and thus allow such binding to be detected) but not to significantly bind to other agents when these conditions are maintained. By way of example, the methods of the present disclosure may be performed under conditions that allow the antibodies of the present disclosure to bind to one or more lungworm antigens present in a particular sample (and thus allow detection of such binding), but not to significantly bind to any hookworm, roundworm, whipworm, heartworm, or other lungworm antigens that may be present in the sample.

[0035] As another example, a polypeptide of the present disclosure is said to be "specific for," "specifically binds," and "stably binds" to a lungworm antibody whenever the antibody is capable of recognizing and binding to these lungworm antigenic polypeptides of the present disclosure with greater affinity than any other antigens derived from parasitic helminths other than lungworms. Such binding specificity can be determined using methods well known in the art, such as ELISA or radioimmunoassay (RIA). Based on the information observed regarding the binding specificity of a particular composition of the present disclosure, the method of the present disclosure can be carried out under conditions that allow the composition to bind to one or more specific agents (and thus allow such binding to be detected) but not to significantly bind to other agents when these conditions are maintained. By way of example, the methods of the present disclosure may be performed under conditions that allow the antigenic polypeptides of the present disclosure to bind to one or more lungworm antibodies present in a particular sample (and thus allow detection of such binding), but not to significantly bind to any hookworm, roundworm, whipworm, heartworm antigens, or other lungworms other than A. abstrusus that may be present in the sample.

[0036] "Detection of lungworms" refers to detection of one or more lungworm-specific products of A.abstrusus, or one or more lungworm antigens or lungworm antibodies, including, for example, one or more of the polypeptides, antibodies, and nucleic acids of the present disclosure.The presence of one or more such lungworm products in a sample from a mammal indicates that the mammal has an A.abstrusus infection, regardless of whether any whole lungworm organisms or their eggs are also present in the sample.Conversely, in the absence of one or more such lungworm products, a sample from a mammal indicates that the mammal does not have an A.abstrusus infection.

[0037] "Amino acid" refers to naturally occurring and synthetic amino acids. Amino acid residues are abbreviated as follows: alanine is A or Ala; arginine is R or Arg; asparagine is N or Asn; aspartic acid is D or Asp; cysteine ​​is C or Cys; glutamic acid is E or Glu; glutamine is Q or Gln; glycine is G or Gly; histidine is H or His; isoleucine is I or Ile; leucine is L or Leu; lysine is K or Lys; methionine is M or Met; phenylalanine is F or Phe; proline is P or Pro; serine is S or Ser; threonine is T or Thr; tryptophan is W or Trp; tyrosine is Y or Tyr; valine is V or Val. Unless otherwise specified herein, X or Xaa represents any amino acid. Other valid amino acids include, but are not limited to, 4-hydroxyproline and 5-hydroxylysine. In all cases, the amino acid sequences of polypeptides described or otherwise referred to herein are presented in conventional form, with the left-most or first amino acid residue of the sequence designated as the N-terminal residue and the right-most or last amino acid residue of the sequence designated as the C-terminal residue.

[0038] A "conservative variant" of any particular nucleic acid sequence includes any sequence having one or more degenerate codon substitutions relative to the particular nucleic acid sequence, any sequence having one or more nucleotide substitutions, insertions, and deletions from the particular nucleic acid sequence, as well as the complementary sequence of the particular nucleic acid and conservative variants of the complementary sequence. A conservative variant of a particular nucleic acid sequence preferably has at least about 85% identity, more preferably at least about 90% identity, and even more preferably at least about 95-99% identity to the particular nucleic acid sequence. Conservative variants of a particular nucleic acid sequence may be artificially synthesized or may be isolated in their natural form from organisms, including organisms that are lungworms, such as A. abstrusus.

[0039] A "conservative variant" of any particular polypeptide sequence is any polypeptide having an amino acid sequence that differs from that of the particular polypeptide such that an antibody of the present disclosure raised against the particular polypeptide is capable of specifically binding to the variant polypeptide, but that still retains the specific binding properties of the particular polypeptide. Thus, for example, a conservative variant of a particular polypeptide may have one or more amino acid substitutions, deletions, additions, and insertions relative to the particular polypeptide. For example, a conservative variant of a particular polypeptide may have 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less conservative amino acid substitutions relative to the particular polypeptide. A conservative variant of a particular polypeptide preferably, but not necessarily, has at least about 80% identity, more preferably at least about 90% identity, and even more preferably at least about 91-99% identity to the particular polypeptide. The percent identity of any subject nucleic acid or amino acid sequence (e.g., any of the polypeptides described herein) compared to another "target" nucleic acid or amino acid sequence may be determined as follows. First, the target nucleic acid sequence or target amino acid sequence of the present disclosure can be compared and aligned to a nucleic acid sequence or amino acid sequence of interest using the BLAST 2 Sequences (Bl2seq) program, a standalone version of BLASTZ that contains BLASTN and BLASTP (e.g., version 2.0.14). The standalone version of BLASTZ can be obtained at www.ncbi.nlm.nih.gov. Instructions explaining how to use BLASTZ, specifically the Bl2seq program, can be found in the "readme" file that accompanies BLASTZ. The program is also described in detail by Karlin et al. (1990), Proc. Natl. Acad. Sci., 87:2264; Karlin et al. (1990), Proc. Natl. Acad. Sci., 90:5873; and Altschul et al. (1997), Nucl. Acids Res., 25:3389.

[0040] Bl2seq performs comparison of subject and target sequences using either the BLASTN algorithm (used to compare nucleic acid sequences) or the BLASTP algorithm (used to compare amino acid sequences). Typically, when performing amino acid sequence alignment, the following default parameters of the BLOSUM62 scoring matrix are used: gap existence cost=11 and extension cost=1, word size=3, expectation=10, cost per residue=1, and lambda ratio=0.85. The output file contains the alignment regions that have homology between the target and subject sequences. Once aligned, the length is determined by counting the number of consecutive nucleotides or amino acid residues (i.e., excluding gaps) from the target sequence that align with the sequence from the subject sequence, starting at any matching position and ending at any other matching position. A matching position is any position where an identical nucleotide or amino acid residue is present in both the target and subject sequences. To maximize sequence alignment between structurally conserved domains (eg, alpha helices, beta sheets, and loops), gaps of one or more residues may be inserted into the target or subject sequence.

[0041] The percent identity over a particular length is determined by counting the number of matching positions over this particular length, dividing this number by the length, and multiplying the resulting value by 100. For example, if (i) a 500 amino acid target sequence is compared to the subject amino acid sequence, (ii) the Bl2seq program presents 200 amino acids from the target sequence aligned with a region of the subject sequence in which the first and last amino acids of this 200 amino acid region are matched amino acids, and (iii) the number of matches over these aligned 200 amino acids is 180, then the 500 amino acid target sequence contains a length of 200 and 90% (i.e., 180 / 200×100=90) sequence identity over this length. It will be understood that the target nucleic acid sequence or target amino acid sequence that aligns with the subject sequence can result in many different lengths, with each length having its own identity percent. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 will round down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 will round up to 78.2. It is also noted that this length value will always be an integer.

[0042] Conservative variants of a particular polypeptide sequence may be artificially synthesized or may be isolated in their natural form, for example, from organisms including lungworm organisms such as A. abstrusus. One skilled in the art will also recognize that these variants include, but are not limited to, those having one or more substitutions of a basic amino acid residue, one or more substitutions of an acidic amino acid residue, one or more substitutions of a polar amino acid residue, one or more substitutions of a hydrophobic amino acid residue, one or more substitutions of an aromatic amino acid residue, and one or more substitutions of a small amino acid residue. ("Basic" amino acid residues are K, R, and H. "Acidic" amino acid residues are D and E. "Polar" amino acid residues are N and Q. "Hydrophobic" amino acids are I, L, and V. "Aromatic" amino acid residues are F, Y, and W. "Small" amino acids are G, S, A, T, and M.)

[0043] As used herein, an "epitope" is a portion of an antigenic polypeptide that reacts with serum derived from an A. abstrusus-infected mammal (i.e., an epitope is specifically bound by one or more antibodies in such serum). Epitopes of the polypeptides described in this application may generally be identified using methods known to those of skill in the art, such as those summarized in Paul, "Fundamental Immunology", 3rd ed., pp. 243-247 (Raven Press, 1993), and references cited therein. For example, polypeptides derived from native lungworm antigens, produced by recombinant or chemical synthetic methods, may be screened for the ability to react with pooled sera obtained from A. abstrusus-infected mammals. Suitable assays for assessing reactivity with lungworm-infected sera, such as enzyme-linked immunosorbent assays (ELISAs), are described in more detail below and in Harlow and Lane, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988. An epitope of a polypeptide is a portion that reacts with such antisera at a level substantially similar to the reactivity of the full-length polypeptide, in other words, an epitope can elicit at least about 80%, and preferably at least about 100%, of the response elicited by the full-length polypeptide in an antibody binding assay (e.g., ELISA).

[0044] Combinatorial polypeptides are disclosed that contain epitopes from multiple antigenic polypeptides. A "combinatorial polypeptide" is a polypeptide in which epitopes from different polypeptides or variants thereof are connected into a single amino acid chain, for example, via peptide bonds. The amino acid chain thus formed may be linear or branched. The epitopes may be connected directly (i.e., without intervening amino acids) or by a linker sequence (e.g., Gly-Cys-Gly) that does not significantly alter the antigenic properties of the epitopes. Polypeptide epitopes may also be linked through non-peptide bonds, such as heterobifunctional or homobifunctional agents that chemically or photochemically couple specific functional groups on the polypeptide epitopes, such as through amino, carboxyl, or sulfhydryl groups. Bifunctional agents that may be usefully employed in the combinatorial polypeptides of the present disclosure are well known to those skilled in the art. Epitopes may also be linked by complementary ligand / antiligand pairs, such as avidin / biotin, in which one or more epitopes are linked to a first member of the ligand / antiligand pair, which is then bound to the complementary member of the ligand / antiligand pair, either in solution or in solid phase. A combination polypeptide may contain multiple epitopes of the polypeptides described herein and / or may contain epitopes of one or more other lungworm antigens. Nucleic Acids and Polypeptides of the Disclosure In an attempt to identify compositions that can be used to confirm the presence or absence of lungworms in mammalian samples, a cDNA library of lambda ZapII was constructed using RNA from L1 larvae of Aelurostrongylus abstrusus. The library was screened with experimentally infected cat serum samples, and several positive clones were identified. These positive clones were excised as plasmids and the inserts were sequenced. The DNA sequences of the positive clones were analyzed in the NCBI database. As a result of these efforts, a 1020 nucleotide cDNA sequence (SEQ ID NO:1) from A. abstrusus was deduced (this sequence is shown in FIG. 1A and identified herein as SEQ ID NO:1). A BLAST search performed using SEQ ID NO:1 indicated that a portion of this sequence was similar, but not identical or substantially identical, to a nucleic acid sequence predicted to encode a portion of VAL (venom allergen / ancylostoma secreted protein-like) 10. ATG is the start codon of the nucleotide sequence, and TAG is the stop codon of the nucleotide sequence. The first 60 nucleotides of the sequence (encoding 20 amino acids) represent the native signal sequence of the secreted protein of A. abstrusus according to the SignalP program.

[0045] Analysis of the sequence corresponding to SEQ ID NO:1 indicated that the sequence contains a large ORF. Specifically, as shown in FIG. 1B, the large ORF of SEQ ID NO:1 corresponds to nucleotides 1004 to 1960 of SEQ ID NO:1 and contains the following amino acid sequence: NDEMDENNPVALPGKSNVSEPCQTTTSNPIHPTTGEQLETTTGKPRRTTKGRILKTTTVKPIKTTKGKKLKTTKGKLLKTTTVKPFKTTKGKPLKTTTKEPFQSTTGGPVGPTTDRCKLNNGMNDQLRDIMLNDHNTLRSLAAKGLAENPLGTNGRAPKAARMLKMVYDCNVEKTAMIHAKKCVFEHSKGRKDTGENVWVMWPAQKNLTEMATRASKSWFDELKKFGVPPDNILTEGLWNRPKMAIGHYTQMVWEGSYKLGCGVATCSDKTLIVCQYSPAGNYIGSIIYAIGEPCKTDEDCKCEGCKCSRDEALCIKPN (SEQ ID NO: 2) It is predicted that the polypeptides of the present disclosure will encode a polypeptide having the following structure: The polypeptides of the present disclosure may be encoded by a nucleic acid having a nucleotide sequence corresponding to all or a portion of SEQ ID NO: 1, or all or a portion of any conservative variant of these sequences. It is therefore understood that the amino acid sequence of the polypeptides of the present disclosure may be variable.

[0046] For example, a polypeptide of the disclosure can have an amino acid sequence corresponding to all or a portion of SEQ ID NO:2, or to all or a portion of any conservative variant of SEQ ID NO:2.

[0047] In one embodiment, the polypeptide of the present disclosure, identified as TDX1557, has the following amino acid sequence: [ka] has.

[0048] The 319 amino acid residues between the IgG-like signal sequence beginning with the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:3 and the HIS tag beginning with amino acid residue 341 represent amino acid residues 1-319 of SEQ ID NO:2. As described in the Examples section contained herein, the IgG-like signal sequence beginning with the N-terminal methionine added to the N-terminus of the polypeptide of SEQ ID NO:2, as well as the HIS tag and epitope tag added to the C-terminus of the polypeptide of SEQ ID NO:2, were artificially added to this polypeptide by performing standard cloning methods. As also described throughout the Examples section, antibodies raised against the polypeptide corresponding to SEQ ID NO:3 were useful for detecting A. abstrusus antigens. The polypeptide of SEQ ID NO:3 is interchangeably referred to as 2M2, TDX1557, recombinant TDX1557, or rTDX1557.

[0049] In another embodiment, a truncated form of the polypeptide of SEQ ID NO:2 of the present disclosure is provided. A schematic representation of a truncated form of the polypeptide of SEQ ID NO:2 is shown in Figure 16B. In one embodiment, a truncated form of the polypeptide of SEQ ID NO:2 has the following sequence: [ka] has.

[0050] The 197 amino acid residues between the IgG-like signal sequence beginning with the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:4 and the HIS tag beginning with amino acid residue 221 represent a portion of the polypeptide of SEQ ID NO:2, specifically, amino acid residues 123-319 of SEQ ID NO:2. As described in the Examples section contained herein, the IgG-like signal sequence beginning with the N-terminal methionine added to the N-terminus of the fragment of the polypeptide of SEQ ID NO:2, as well as the HIS tag and epitope tag added to the C-terminus of the portion of the polypeptide of SEQ ID NO:2, were artificially added by performing standard cloning methods. As also described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:4 was useful for detecting lungworm antibodies present in a mammal infected with A. abstrusus in a sample (e.g., serum). The polypeptide of SEQ ID NO:4 is interchangeably referred to as the polypeptide 2M2T2, TDX1637, or T2.

[0051] In another embodiment, a truncated form of the polypeptide of SEQ ID NO:2 has the following sequence: [ka] has.

[0052] The 196 amino acid residues between the IgG-like signal sequence beginning with the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:5 and the HIS tag beginning with amino acid residue 221 represent a portion of the polypeptide of SEQ ID NO:2, specifically, amino acid residues 1-196 of SEQ ID NO:2. As described in the Examples section contained herein, the IgG-like signal sequence beginning with the N-terminal methionine added to the N-terminus of the portion of the polypeptide of SEQ ID NO:2, as well as the HIS tag and epitope tag added to the C-terminus of the portion of the polypeptide of SEQ ID NO:2, were artificially added by performing standard cloning methods. As also described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:5 was useful for detecting lung nematode antibodies present in a mammal infected with A. abstrusus in a sample (e.g., serum). The polypeptide of SEQ ID NO:5 is interchangeably referred to as the polypeptide 2M2T3, TDX1674, or T3.

[0053] In one embodiment, a truncated form of the polypeptide of SEQ ID NO:2 has the following sequence: [ka] has.

[0054] The 130 amino acid residues between the IgG-like signal sequence beginning with the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:6 and the HIS tag beginning with amino acid residue 155 represent a portion of the polypeptide of SEQ ID NO:2, specifically, amino acid residues 190-319 of SEQ ID NO:2. As described in the Examples section contained herein, the IgG-like signal sequence beginning with the N-terminal methionine added to the N-terminus of the portion of the polypeptide of SEQ ID NO:2, as well as the HIS tag and epitope tag added to the C-terminus of the portion of the polypeptide of SEQ ID NO:2, were artificially added by performing standard cloning methods. As also described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:6 was useful for detecting lung nematode antibodies present in a mammal infected with A. abstrusus in a sample (e.g., serum). The polypeptide of SEQ ID NO:6 is interchangeably referred to as the polypeptide 2M2_T4, TDX1675, or T4.

[0055] In another embodiment, a polypeptide of the disclosure can have an amino acid sequence corresponding to a portion of SEQ ID NO: 2, or a portion of any conservative variant of SEQ ID NO: 2. In a particular embodiment, the peptide has the following amino acid sequence: C10 MVYDCNVEKTAMIHA (SEQ ID NO: 7) C11 NVEKTAMIHAKKCVF (SEQ ID NO: 8) C12 AMIHAKKCVFEHSKG (SEQ ID NO: 9) D01 KKCVFEHSKGRKDTG (SEQ ID NO: 10) D02 EHSKGRKDTGENVWV (SEQ ID NO: 11) D03 RKDTGENVWVMWPAQ (SEQ ID NO: 12) D04 ENVWVMWPAQKNLTE (SEQ ID NO: 13) D05 MWPAQKNLTEMATRA (SEQ ID NO: 14) D06 KNLTEMATRASKSWF (SEQ ID NO: 15) D07 MATRASKSWFDELKK (SEQ ID NO: 16) D08 SKSWFDELKKFGVPP (SEQ ID NO: 17) has.

[0056] These peptides may be useful for designing antigenic combination polypeptides for detecting antibodies in a sample, eliciting antibodies against lungworm antigens, and / or detecting A.abstrusus antibodies in a sample. For example, based on the alignment of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, which are the polypeptide sequences corresponding to D6, D7, and D8, which are the polypeptides shown in Figure 13, a combination polypeptide of SEQ ID NO:18 was constructed (labeled D678 or D678 combo).

[0057] D678 combo (SEQ ID NO: 18), which is CPAQKNLTEMATRASKSWFDELKKFG

[0058] It is also envisioned that any one or more of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 may be a small portion of a larger polypeptide sequence and thus may represent a partial sequence of one or more proteins normally expressed in, for example, A. abstrusus, or may represent one or more polypeptide sequences artificially fused to one or more of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. Those skilled in the art will recognize that a wide variety of techniques exist for artificially fusing two or more polypeptide fragments together.

[0059] It is still further contemplated that a polypeptide of the present disclosure may comprise more than one of the sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. 。It is also envisioned that a polypeptide of the disclosure may comprise multiple polypeptide fragments corresponding to one or more of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. For example, a polypeptide of the disclosure may be formed by multiple polypeptide fragments corresponding to SEQ ID NO:6 fused together. In another example, a polypeptide of the disclosure may be formed by multiple polypeptide fragments corresponding to SEQ ID NO:6 and multiple polypeptide fragments corresponding to SEQ ID NO:7 fused together, in any combination.

[0060] Although the various polypeptides of the present disclosure have been expressed and isolated by specific recombinant methods or chemically synthesized (as described in the Examples section contained herein), one of skill in the art will recognize that any of the polypeptides of the present disclosure may be prepared and / or isolated by utilizing any one or more of a wide variety of techniques (see, e.g., Sewald and Jakubke, "Peptides: Chemistry and Biology", Wiley Publishing (2002); "Peptide Synthesis and Applications" ("Methodes in Molecular Biology"), ed. Howl, Humana Press (2005); Jones, "Amino Acid and Peptide Synthesis", Oxford University Press (2002), each of which is incorporated herein by reference in its entirety). These techniques include techniques that may be performed to isolate naturally occurring polypeptides having amino acid sequences corresponding to SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, as well as any naturally occurring variants of these polypeptides. These techniques further include techniques that may be performed to artificially create polypeptides having amino acid sequences corresponding to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, as well as any conservative variants of these polypeptides. Such variants may be created, for example, by utilizing any one or more mutagenesis techniques or by direct synthesis.

[0061] The polypeptide of the present disclosure is useful as a reagent for detecting the presence of anti-A.abstrusus antibodies in a sample. When immobilized on a substrate and contacted with a sample obtained from a mammal, the immobilized polypeptide can specifically bind to anti-A.abstrusus antibodies, but cannot specifically bind to any antibodies from hookworms, roundworms, or heartworms that may be present in the sample. The polypeptide of the present disclosure may be suitable for use only to capture one or more lungworm antibodies, or may be suitable for use only to detect one or more lungworm antibodies, but is more preferably suitable for use to capture and detect one or more lungworm antibodies.

[0062] The polypeptides are also capable of eliciting an immune response in a host animal that is exposed to these polypeptides to generate one or more of the antibodies of the present disclosure. Regardless of the technique by which it is derived, the polypeptides of the present disclosure are preferably prepared in a substantially pure form when used as reagents for the purpose of raising antibodies. Preferably, these polypeptides are at least about 80% pure, more preferably at least about 90-95% pure, and even more preferably at least about 99% pure. Although exemplary techniques for eliciting an immune response in a host organism and isolating antibodies from these host organisms are described herein, it is to be understood that the present disclosure is not limited to these techniques. Those skilled in the art will recognize that there are multiple techniques for achieving this same goal without departing from the scope and spirit of the present disclosure.

[0063] Antibodies of the present disclosure The present disclosure further includes antibodies and antigen-binding fragments thereof that are raised against and specifically bind to all or a portion of one or more polypeptides of the present disclosure, and also includes compositions comprising said antibodies and antigen-binding fragments. When contacted with a sample obtained from a mammal, these antibodies and antigen-binding fragments are capable of specifically binding to A.abstrusus antigens present in the sample, but are not capable of specifically binding to any antigens from hookworms, roundworms, or heartworms that may be present in the sample. The antibodies of the present disclosure may be suitable for use only to capture one or more lungworm antigens, or may be suitable for use only to detect one or more lungworm antigens, but are more preferably suitable for use to capture and detect one or more lungworm antigens.

[0064] The antibodies of the disclosure can belong to any antibody class, including, for example, IgG, IgM, IgA, IgD, and IgE, and can be prepared by any of a wide variety of techniques known to those of skill in the art (see, e.g., Dean, Methods Mol. Biol., 80:23-37 (1998); Dean, Methods Mol. Biol., 32:361-79 (1994); Baileg, Methods Mol. Biol., 32:381-88 (1994); Gullick, Methods Mol. Biol., 32:389-99 (1994); Drenckhahn et al., Methods Mol. Biol., 32:389-99 (1994); each of which is incorporated herein by reference in its entirety). See Cell. Biol., 37:7-56 (1993); Morrison, Ann. Rev. Immunol., 10:239-65 (1992); Wright et al., Crit. Rev. Immunol., 12:125-68 (1992); Harlow and Lane, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory (1988); and "Making and Using Antibodies: A Practical Handbook", Howard and Kaser, eds., CRC Press (2006)).

[0065] In one technique, the polypeptide of the present disclosure is introduced into a host animal, such as, for example, rabbit, mouse, rat, guinea pig, goat, pig, cow, sheep, donkey, dog, cat, chicken, or horse. Enhanced immune response may be induced in the host animal by associating the polypeptide with a carrier and / or by exposing the host to an adjuvant, but it is understood that the present disclosure does not require that the polypeptide be associated with a carrier or that the host be exposed to an adjuvant. Exemplary carriers that can be used for this purpose are bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Exemplary adjuvants include Freund's complete or incomplete adjuvant, and MDL-TDM adjuvant. Regardless of whether the polypeptide is associated with such a carrier or whether the host is exposed to an adjuvant, the host animal can be immunized with an optional booster dose and then bled one or more times.The polyclonal antibody that specifically binds to the polypeptide can then be purified from the antiserum obtained from one or more blood draws.Such purification can be achieved, for example, by utilizing affinity chromatography methods that involve associating the polypeptide with a solid support.Such affinity chromatography methods are well known to those skilled in the art.

[0066] In one embodiment, the antibody of the present disclosure is an antibody raised in rabbits by immunizing a host animal with a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 3 (hereinafter, this particular antibody is referred to as "anti-TDX1557"). In the Examples section contained herein, a specific technique for producing and isolating anti-TDX1557 pAB is described, but one skilled in the art will recognize that the production and isolation of anti-TDX1557 pAB, or any other antibody of the present disclosure, is not limited to this specific technique.

[0067] In another embodiment, the antibodies of the disclosure are raised in a host against one or more polypeptides having an amino acid sequence that is a conservative variant of the sequence corresponding to SEQ ID NO: 3. In some other embodiments ...2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 truncation), SEQ ID NO: 5 (T3 truncation), SEQ ID NO: 6 (T4 truncation), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 peptide), SEQ ID NO: 19 (d78 peptide), SEQ ID NO: 20 (d88 peptide), SEQ ID NO: 21 (d98 peptide), SEQ ID NO: 22 (d10 peptide), SEQ ID NO: 23 (d11 peptide), SEQ ID NO: 24 (d12 peptide), SEQ ID NO: 25 (d13 peptide), SEQ ID NO: 26 (d14 peptide), SEQ ID NO: 27 (d15 peptide), SEQ ID NO: 28 (d16 peptide), SEQ ID NO: 29 (d17 peptide), SEQ ID combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino-extended), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: No. 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), SEQ ID NO: 115 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117), in which (a) the amino acids W and F at positions 4 and 5, respectively, are retained and at least one X at positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made at positions 1, 2, and 3;(c) the amino acids W and F at positions 4 and 5, respectively, are retained, and X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid; (d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or one or more polypeptides having an amino acid sequence that is a conservative variant of any of these sequences;

[0068] In another embodiment, the antibody of the disclosure is selected from the group consisting of SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 truncate), SEQ ID NO:5 (T3 truncate), SEQ ID NO:6 (T4 truncate), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d40 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO:39 (d16 peptide), SEQ ID NO:40 combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:115 (D8M 9), one or more polypeptides having an amino acid sequence corresponding to SEQ ID NO:116 (D8M10), or XXXWF (SEQ ID NO:117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid, or an antibody that specifically binds to an antigenic portion thereof;

[0069] In other further embodiments, the antibodies of the disclosure are selected from the group consisting of SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 truncate), SEQ ID NO:5 (T3 truncate), SEQ ID NO:6 (T4 truncate), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d40 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO:39 (d16 peptide), SEQ ID NO:4 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino-extended), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), SEQ ID NO: 115 (D8M9), SEQ ID NO: No. 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in which: (a) the amino acids W and F at positions 4 and 5, respectively, are retained and at least one X at positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) the amino acids W and F at positions 4 and 5, respectively, are retained and X at positions 1 and 3 is independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is replaced with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid;

[0070] It should also be understood that the antibodies of the present disclosure may be polyclonal or monoclonal antibodies, single chain antibodies (scFv), chimeric antibodies, and fragments thereof. Monoclonal antibodies specific to a polypeptide of interest can be obtained and purified, for example, by preparing a cell line that produces an antibody with the desired specificity against the polypeptide of interest. This type of cell line can be derived from a specific type of cell (e.g., spleen cells) that is isolated from a host animal previously immunized with a polypeptide as previously described. In such a case, these cells can then be immortalized by fusing them with myeloma cells, for example, by carrying out any one of a wide variety of fusion methods known to those skilled in the art. In one exemplary technique, cells from an immunized host animal are co-incubated with their fusion partner, e.g., myeloma cells, for a short period in the presence of detergent and then plated on a medium that supports the growth of hybrid cells (but not the growth of the myeloma fusion partner). Such selection can be accomplished, for example, by using hypoxanthine, aminopterin, and thymidine (HAT). Once hybrid cells have begun the selection process, perhaps after one or two weeks of selection, single hybrid colonies (and their supernatants) are examined for their ability to bind to one or more polypeptides against which the host animal has been immunized. Hybrid colonies with optimal binding specificity will represent the best candidates from which monoclonal antibodies can be isolated. These monoclonal antibodies can be isolated, for example, directly from the supernatant (i.e., medium) in which these colonies are grown, by utilizing any one of a wide variety of techniques known to those of skill in the art.

[0071] The antibodies of the present disclosure can also be single chain antibodies (scFv), or antigen-binding fragments of antibodies. An antigen-binding fragment of an antibody is a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, where the portion does not contain the heavy chain constant domain of the Fc region of the intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, and F(ab')2 fragments. v In addition to production and purification from animal or mammalian cells, antibodies, antibody fragments, or non-antibody scaffolds can also be selected based on a variety of in vitro methods, including phage display, ribosome display, or bacterial display.

[0072] Antibodies, including secondary antibodies, can be labeled with any type of label known in the art, including, for example, fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, colloidal particle labels, radioisotope labels, and bioluminescent labels. In various embodiments of the present disclosure, one or more of the antibodies of the present disclosure are labeled with enzymes, colloidal particles, radionuclides, or fluorophores. Particulate labels can be, for example, colored latex particles, dye sols, or gold sols conjugated to antibodies.

[0073] Methods, devices, and kits of the present disclosure Devices and kits of the present disclosure In one aspect, the disclosure is a device for detecting A. abstrusus infection in a mammal, such as, for example, a dog, cat, pig, cow, or human. The device is arranged to aid in the detection of the presence or absence of A. abstrusus antibodies (or the presence or absence of A. abstrusus antigens in a sample using the antibodies of the disclosure) from a mammal that may also be infected with one or more other helminth parasites, including hookworms, roundworms, whipworms, and heartworms, using the polypeptides of the disclosure.

[0074] In one embodiment, the device comprises a solid support, in which one or more polypeptides (or antibodies) of the present disclosure are immobilized on the solid support. The solid support can be, but is not limited to, the well of a microtiter plate, or the inner bottom surface of a substrate that is incorporated as part of a lateral flow device, for example. An exemplary microtiter plate is the Immulon 1B 96-well plate (commercially available from Thermo Scientific of Milford, MA), but it is understood that those skilled in the art will recognize that a wide variety of other microtiter plates other than the Immulon 1B 96-well plate allow for the immobilization of polypeptides (or antibodies) thereon, and thus are suitable for preparing the solid support of the present disclosure.

[0075] An exemplary lateral flow device is the lateral flow device described in U.S. Patent No. 5,726,010, which is incorporated herein by reference in its entirety. A device for performing a lateral flow assay can be a SNAP® device, commercially available from IDEXX Laboratories, Inc., Westbrook, ME. However, it is to be understood that those skilled in the art will recognize that a wide variety of other lateral flow devices other than the SNAP® device, or other wide variety of lateral flow devices described by U.S. Patent No. 5,726,010, are also suitable for use as the device of the present disclosure, since they allow for the immobilization of a polypeptide (or antibody) thereon. These devices can include, for example, lateral flow devices using colloidal gold technology.

[0076] The polypeptides or antibodies used in the devices of the present disclosure may be immobilized on a solid support by any method known in the art, including, for example, methods of attaching antibodies to a solid support, either covalently or non-covalently, directly or indirectly. Thus, although the antibodies or polypeptides may be attached to the solid support by physical adsorption (i.e., without the use of chemical linkers), it is also plausible that the antibodies or polypeptides may be immobilized to the solid support by any chemical binding method (i.e., using chemical linkers) that is readily known to those skilled in the art.

[0077] It should also be understood that the solid support can be any suitable material for immobilizing an antibody or polypeptide of the disclosure. For example, the solid support can be a bead, particle, tube, well, probe, dipstick, pipette tip, slide, fiber, membrane, paper, natural and modified cellulose, polyacrylamide, agarose, glass, polypropylene, polyethylene, polystyrene, dextran, nylon, amylase, plastic, magnetite, or any other suitable material readily known to one of skill in the art.

[0078] The device may optionally include one or more labeled antigen or antibody capture reagents that may be mixed with a sample from a mammal prior to application to the device of the present disclosure. When a labeled capture antigen or antibody reagent is incorporated, the labeled antigen or antibody capture reagent may or may not be deposited or dried on the solid surface of the device. An "antigen capture reagent" refers to any compound that is specific for one or more antigens of interest. The labeled antigen capture reagent, whether added to the mammalian sample or pre-deposited on the device, may be, for example, a labeled antibody specific for an A.abstrusus antigen, including but not limited to the antibody of the present disclosure. In one example, anti-TDX1557 pAB conjugated with horseradish peroxidase may be used as a labeled antigen capture reagent. An "antibody capture reagent" refers to any compound that specifically binds to one or more antibodies of interest. The labeled antibody capture reagent, whether added to the mammalian sample or pre-deposited on the device, can be, for example, a labeled polypeptide specific for A. abstrusus antibodies, including, but not limited to, the polypeptides of the present disclosure. In one example, TDX1557, a recombinant protein conjugated with horseradish peroxidase, can be used as a labeled antibody capture reagent.

[0079] The device may also optionally include liquid reagents that either transfer (such as, for example, if the device is a SNAP® device) or otherwise facilitate removal (such as, for example, if the device includes a microtiter plate) of unbound materials (e.g., unreacted portions of a mammalian sample, such as, for example, unreacted portions of serum or fecal extract, and unbound antigen or antibody capture reagent) from the reaction zone (solid phase). The liquid reagent may be a wash reagent and used only to remove unbound materials from the reaction zone, or it may include a detection reagent and be used both to remove unbound materials and to facilitate detection of the antigen or antibody. For example, in the case of an antigen or antibody capture reagent conjugated to an enzyme, the detection reagent includes a substrate that, upon reaction with the enzyme-antibody conjugate, provides a detectable signal in the reaction zone (solid phase). Alternatively, in the case of labeled antigen or labeled antibody capture reagents conjugated to radioactive, fluorescent, or light-absorbing molecules, the liquid reagent acts only as a wash fluid to wash away unbound labeled reagent, thereby facilitating detection of complex formation in the reactive zone.

[0080] The liquid reagent may further comprise a limited amount of an "inhibitor," i.e., a substance that prevents the development of a detectable end-product, the limited amount being defined as an amount of inhibitor sufficient to prevent the development of the end-product until most or all of the excess unbound material has been displaced from the second region, at which point a detectable end-product is produced.

[0081] The device of the present disclosure may also include a variety of binding reagents immobilized at a location significantly different from one or more antigen capture reagents or antibody capture reagents. For example, an immune reagent (antibody, antigen or polypeptide) that recognizes the species-specific (e.g., A. abstrusus-specific) antibody portion of the labeled antibody or labeled antigen capture reagent, or the enzyme portion of the enzyme-labeled reagent, may be incorporated as a positive control to evaluate the effectiveness of the reagent in the device. For example, the positive control may be an anti-horseradish peroxidase antibody, for example, raised in a goat or mouse. In addition, an antibody isolated from a non-immune member of the species from which the reagent, for example, the antibody portion of the antigen-antibody complex, is derived, may be incorporated as a negative control to evaluate the specificity of immune complex (i.e., antigen-antibody complex) formation.

[0082] In addition to being designed to detect A. abstrusus in a mammalian sample, the device of the present disclosure may also be designed to optionally allow one or more other diagnostic tests to be performed. For example, the solid support may also include reagents for the detection of one or more lungworm parasites other than A. abstrusus, one or more non-lungworm parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria. A reagent for the detection of one or more non-lungworm parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria may be, for example, one or more antibodies, or one or more antigens, which are recognized by an antibody specific for one or more non-lungworm parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria.

[0083] In one embodiment, the device of the disclosure is a microtiter plate containing a plurality of wells, where each well contains a solid support having immobilized thereon a polypeptide of the disclosure, e.g., recombinant TDX1557, a conservative variant of TDX1557, or a truncated form of TDX1557.

[0084] The plate can be used with the method of the present disclosure to detect the lung nematode A.abstrusus in a mammalian sample. Specifically, lung nematode infection can be diagnosed in a mammal by detecting one or more lung nematode antibodies with the polypeptide of the present disclosure, such as recombinant TDX1557, conservative variants of TDX1557, or truncated forms of TDX1557, immobilized on a solid support. In one embodiment, the antibody detected is a lung nematode antibody. A "lung nematode antibody" is an antibody against any one or more products of lung nematode that are present in a sample, such as a serum sample, and can specifically and stably bind to recombinant TDX1557, conservative variants of TDX1557, or truncated forms of TDX1557. Thus, the lung nematode antibody can be an antibody against whole lung nematode, lung nematode eggs, lung nematode fragments, or products, or combinations thereof, secreted, excreted, or discharged from lung nematodes. Pulmonary nematode antigens may include any one of the polypeptides of the present disclosure, such as, for example, polypeptides having amino acid sequences corresponding to SEQ ID NO:2-SEQ ID NO:17, polypeptides having amino acid sequences that are conservative variants of these sequences, and / or antigenic fragments of any such polypeptides.

[0085] The present disclosure further includes an assay kit (e.g., an article of manufacture) for detecting the lung nematode A.abstrusus in a mammalian sample. Thus, the kit may include one or more devices and / or compositions of the present disclosure. For example, the kit may include a polypeptide or antibody of the present disclosure, and a means for determining the binding of an antigenic polypeptide of the present disclosure to lung nematode antibodies in a sample, or the binding of an antibody of the present disclosure to lung nematode antibodies in a sample. In a particular example, such a kit includes a device immobilizing a polypeptide of the present disclosure, such as rTDX1557, for example, one or more antibody capture reagents (e.g., the antibody capture reagent is immobilized, but not the labeled antibody capture reagent), and a washing reagent, as well as a detection reagent, and positive and negative control reagents, if desired or appropriate. Other components, such as buffers, controls, etc., known to those skilled in the art, may also be incorporated into such a test kit. The relative amounts of various reagents may be varied to provide concentrations of the reagents in solution that substantially optimize the sensitivity of the assay. In particular, reagents may be provided as dry powders, typically lyophilized, that upon dissolution provide a reagent solution having an appropriate concentration for combination with the sample. The kits may further include instructions for carrying out one or more methods of the present disclosure, including instructions for using any of the devices and / or compositions of the present disclosure incorporated with the kit.

[0086] Methods of the Disclosure The present disclosure further includes a method for detecting the presence or absence of the lung nematode A.abstrusus in a sample using one or more of the devices, kits, and / or compositions of the present disclosure. Thus, the method can be implemented to detect the presence or absence of the lung nematode in a sample, such as a serum sample obtained from a mammal, including but not limited to a dog, a cat, a pig, a cow, or a human. Furthermore, the method can be implemented to detect, for example, roundworms, hookworms, whipworms, and / or heartworms. Furthermore, these methods are useful for confirming the presence or absence of the lung nematode in such a sample, even if the sample contains one or more products from other helminth species, including one or more products from hookworms, whipworms, roundworms, and / or heartworms.

[0087] In one embodiment of the method of the present disclosure, detection of the lung nematode A. abstrusus can be achieved by detecting the presence or absence of one or more lung nematode antibodies in a sample using a polypeptide of the present disclosure, such as a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 2-SEQ ID NO: 18, as well as, for example, fragments and / or conservative variants of these sequences. In another embodiment of the present disclosure, detection of the lung nematode can be achieved by detecting the presence or absence of one or more lung nematode antigens in a sample using an antibody of the present disclosure, such as, for example, rTDX1557 pAb. Those skilled in the art will recognize that there are a wide variety of ways to extract and prepare non-fecal samples from a mammal as well. For example, the sample can be a serum sample obtained from blood, a sample obtained by swabbing a mammal, such as blood and associated fluids, such as serum and plasma, fluids and tissues obtained from throat swabs, transtracheal aspirates, endotracheal swabs, bronchoalveolar lavage, pleural effusions, and other materials, for example, obtained from the respiratory tract of a mammal. As yet another example, tissue sections including tissue from the small intestine, large intestine, cecum, colon, rectum, or other tissue of the digestive tract can be obtained by biopsy.

[0088] One embodiment of the method includes contacting a mammalian sample with one or more polypeptides of the present disclosure specific to one or more antibodies against the lung nematode A.abstrusus under conditions that allow for the formation of an antigen / antibody complex, i.e., an immune complex, i.e., an antigenic polypeptide that specifically binds to the lung nematode antibodies present in the sample. Another embodiment of the method includes contacting a mammalian sample with one or more antibodies of the present disclosure specific to one or more lung nematode antigens under conditions that allow for the formation of an antigen / antibody complex, i.e., an antibody that specifically binds to the lung nematode antigen present in the sample. Those skilled in the art are familiar with assays and conditions that can be used to detect the binding of such antigen / antibody complexes. For example, the antigen / antibody complex can be detected using a secondary antibody that binds to the antigen / antibody complex. The formation of a complex between the polypeptide of the present disclosure and the lung nematode antibody in the sample can be detected using any suitable method known in the art.

[0089] Furthermore, the relative amount of antibody-antigen complex formed in one particular reaction can be measured in light of the antibody-antigen complex formed in any other reaction by any method known in the art to achieve this goal. If the test sample is determined to have more antibody-antigen complexes than the control sample, it can be concluded that lungworms are present in the test sample. If this is true, it can be concluded that the mammal from which the test sample was obtained has a lungworm infection. One or both of the conclusions that lungworms are present in the test sample and that the test mammal has a lungworm infection can be made by, for example, a physician at a diagnostic service provider, or by the mammal's caregiver, such as the mammal's veterinarian. If the mammal's caregiver determines (or is otherwise informed of) that the mammal has a lungworm infection, the caregiver places the mammal under a course of treatment optimally designed to specifically remove the lungworms from the mammal, rather than a general infection with nematode parasites. Additionally, the present disclosure may be used to confirm that any animal that has been treated for a lungworm infection has been cleared of the infection.

[0090] The steps of the disclosed method may include applying a mammalian sample to a device of the disclosure that includes an immobilized polypeptide specific for one or more antibodies to the lung nematode A. abstrusus (or an immobilized antibody specific for one or more lung nematode antigens), and detecting the presence or absence of lung nematode antibodies (or antigens) in the sample. The polypeptide specific for an antibody to the lung nematode or the antibody specific for a lung nematode antigen may be directly or indirectly attached to a solid support or substrate, such as a microtiter well, a polypeptide (or antibody) immobilization portion of a SNAP® device, a magnetic bead, a non-magnetic bead, a column, a matrix, a membrane, a fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials, or thermoplastic polymers such as polyethylene, polypropylene, or polyester), a sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or a cast thin film (generally synthetic) composed of nitrocellulose, nylon, polysulfone, or the like. All of these substrate materials may be used in suitable shapes, such as films, sheets, or plates, and suitable inert supports, such as paper, glass, plastic films, or fabrics, may be coated with these substrate materials, or these substrate materials may be bonded or laminated to these suitable inert supports. Suitable methods for immobilizing peptides on solid phases include ionic interactions, hydrophobic interactions, covalent interactions, and the like.

[0091] However, the disclosed method does not require the use of a solid phase or substrate. One skilled in the art will recognize that there are several ways in which the method can be performed to detect the presence or absence of the lung nematode A. abstrusus without the use of a solid phase or substrate. As just one example, an immunoprecipitation method can be performed that does not require the use of a solid phase or substrate.

[0092] In some embodiments of the present disclosure, the antigen / antibody complex is detected when an indicator reagent, such as an enzyme conjugate, bound to the antibody catalyzes a detectable reaction. Optionally, the indicator reagent, which includes a signal generating compound, can be applied to the antigen / antibody complex under conditions that allow for the formation of a detectable antigen / antibody / indicator complex. Optionally, the antibody can be labeled with an indicator reagent prior to the formation of the antigen / antibody complex.

[0093] The formation of antigen / antibody complexes or antigen / antibody / indicator complexes in some of the methods of the present disclosure can be specifically detected by radiometric, colorimetric, fluorometric, photometric, size separation, or precipitation methods. Detection of antigen / antibody complexes can also be achieved by the addition of a secondary antibody coupled to an indicator reagent that contains a signal-generating compound. The indicator reagent that contains a signal-generating compound (label) associated with the polypeptide / antibody complex can be detected using the above methods and can include color developers, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridinium, phenanthridinium, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The choice of a particular label is not critical, but could be capable of producing a signal by itself or together with one or more additional substances.

[0094] The methods of the present disclosure include, but are not limited to, competitive, direct reaction, or sandwich-type assay-based methods, including, but not limited to, ELISA, RIA, immunofluorescence assay (IFA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), and microtiter plate assay (i.e., any assay performed in one or more wells of a microtiter plate). One assay of the present disclosure includes a reversed-phase chromatographic binding assay, which can be performed, for example, by using a SNAP® device. See U.S. Patent No. 5,726,010.

[0095] In some embodiments, the method of the present disclosure facilitates sandwich-type specific binding assay or competitive-type specific binding assay. In sandwich assay, antibody capture reagents are immobilized in reactive zone. These antibody capture reagents can specifically bind to the antibody in the sample examined for lungworm. After the antibody from the sample binds, the antibody capture reagent / antibody complex is detected by any suitable method. For example, the complex can be reacted with a labeled specific binding reagent (e.g., an enzyme-antibody conjugate) and the antibody to be detected (e.g., upon reaction with a substrate).

[0096] In another embodiment of the method of the present disclosure, a competitive assay is carried out. In the competitive assay, the antibody capture reagent is immobilized in the reactive zone and contacted simultaneously with the antibody from the sample and the labeled antibody (e.g., antibody-enzyme conjugate). The amount of label detected in the reactive zone is inversely proportional to the amount of antibody in the sample.

[0097] In some embodiments of the method, a polypeptide that specifically binds to an antibody(ies) against the lungworm A. abstrusus is conjugated to a solid phase or substrate. A sample potentially containing antibodies from the lungworm is added to the substrate. Secondary antibodies that specifically bind to the lungworm antibodies are added. These secondary antibodies may be linked to indicator reagents such as enzyme conjugates. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color may be allowed to develop. The color reaction may be stopped and the color may be quantified, for example, using a spectrophotometer, and / or the color may be subjectively evaluated by the human eye.

[0098] In another embodiment of the method, a polypeptide specific for the antibody(ies) against the lung nematode A.abstrusus is conjugated to a solid phase or substrate. A sample potentially containing lung nematode antibodies is added to the substrate. Second anti-species antibodies that specifically bind to the antibodies against the lung nematode are added. These second antibodies are from a different species than the solid phase antibodies. A third anti-species antibody that specifically binds to the second antibody but not to the solid phase antibody is added. The third antibody may include an indicator reagent, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color may be allowed to develop. The color reaction may be stopped and the color may be quantified, for example, using a spectrophotometer, and / or the color may be subjectively evaluated by the human eye.

[0099] In a specific example, the method of the present disclosure is carried out with a device that is a lateral flow assay device by adding a prepared mammalian sample to the flow matrix of the device in a first region (sample application zone). The prepared sample is transported through the fluid flow path by capillary action to a second region of the flow matrix where a particulate label is present that can bind to the antibody in the sample and form a first complex therewith. The particulate label can be, for example, a colored latex particle, a dye sol, or a gold sol conjugated to an antibody that is specific to an antibody against the lung nematode A. abstrusus. The first complex is transported to a third region of the flow matrix where a polypeptide that specifically binds to the lung nematode antibody is immobilized at a significantly different location. A second complex is formed between the immobilized polypeptide and the first complex. The particulate label that is part of the second complex can be directly visualized by the human naked eye.

[0100] The polypeptide may be an immobilized antibody capture reagent in the reaction zone (solid phase). A second antibody capture reagent, which is a second antibody conjugated to a label, may be added to the sample before the sample is added to the device, or may be incorporated into the device. For example, the labeled antibody capture reagent may be deposited and dried on a fluid flow path that provides fluid communication between the sample application zone and the solid phase. Contact of the labeled antibody capture reagent with the test sample may result in dissolution of the labeled antibody capture reagent.

[0101] In another embodiment of the method, an antibody that specifically binds to one or more antigens from the lung nematode A. abstrusus is conjugated to a solid phase or substrate. A sample potentially containing one or more antigens from the lung nematode is added to the substrate. A secondary antibody that specifically binds to the lung nematode antigen is added. These secondary antibodies may be linked to an indicator reagent, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color may be allowed to develop. The color reaction may be stopped and the color may be quantified, for example, using a spectrophotometer, and / or the color may be subjectively evaluated by the human eye.

[0102] In another embodiment of the method, an antibody specific for one or more antigens from the lung nematode A. abstrusus is conjugated to a solid phase or substrate. A sample potentially containing lung nematode antigens is added to the substrate. A second anti-species antibody is added that specifically binds to the lung nematode antigen. These second antibodies are from a different species than the solid phase antibody. A third anti-species antibody is added that specifically binds to the second antibody but not to the solid phase antibody. The third antibody may include an indicator reagent, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color may be allowed to develop. The color reaction may be stopped and the color may be quantified, for example, using a spectrophotometer, and / or the color may be subjectively evaluated by the human eye.

[0103] In a specific example, the method of the present disclosure is carried out with a device that is a lateral flow assay device by adding a prepared mammalian sample to the flow matrix of the device in a first region (sample application zone). The prepared sample is transported in the fluid flow path by capillary action to a second region of the flow matrix where a particulate label is present that can bind to an antigen in the sample and form a first complex therewith. The particulate label can be, for example, a colored latex particle, a dye sol, or a gold sol conjugated to an antibody specific for a lungworm antigen. The first complex is transported to a third region of the flow matrix where an antibody that specifically binds to a lungworm antigen is immobilized at a significantly different location. A second complex is formed between the immobilized antibody and the first complex. The particulate label that is part of the second complex can be directly visualized by the human naked eye.

[0104] The antibody can be an immobilized antigen capture reagent in the reaction zone (solid phase). A second antigen capture reagent, which is a second antibody conjugated to a label, can be added to the sample before the sample is added to the device or can be incorporated into the device. For example, the labeled antigen capture reagent can be deposited and dried on a fluid flow path that provides fluid communication between the sample application zone and the solid phase. Contact of the labeled antigen capture reagent with the test sample can result in dissolution of the labeled antigen capture reagent.

[0105] In one embodiment of the method of the present disclosure, antibodies to A. abstrusus (or antigens from A. abstrusus) are detected by ELISA. Specific examples of the ELISA methods of the present disclosure are described in the Examples section contained herein. Although the present disclosure is described with respect to these specific ELISA methods, it is to be understood that one of skill in the art will recognize that alternative, additional, or substitute ELISA steps may be used without departing from the basic goal achieved through the method of the present disclosure.

[0106] In another embodiment of the methods of the present disclosure, antibodies to the lungworm A. abstrusus (or antigens from the lungworm A. abstrusus) are detected by using a lateral flow device, such as, for example, a SNAP® device.

[0107] Furthermore, the method of the present disclosure for detecting lungworm infection can be combined with other diagnostic assays to detect the presence of other organisms or conditions. For example, the assay of the present disclosure can be combined with reagents that detect one or more non-lungworm helminth fecal parasites, one or more non-helminth fecal parasites, one or more viruses, one or more fungi, one or more bacteria, one or more blood-borne parasites or occult blood, or combinations thereof. By placing two or more unique binding sites in a single assay device (such as two unique spots on a SNAP® assay device), the present disclosure allows the detection of two or more organisms from a single sample. In one embodiment, there are three unique spots (the spots are antigen- or antibody-binding reagents) to detect past or current infection or infestation from three organisms from a single sample (i.e., the same individual sample is exposed to three capture reagents on a single device). In yet another embodiment, there are four unique spots (the spots being antigen or antibody binding reagents) to detect past or current infection or infestation from four organisms from a single sample (i.e., the same individual sample is exposed to four capture reagents on a single device). However, it should be understood that the same device can contain more than four unique spots and / or allow for the detection of more than four organisms.

[0108] A reagent for detecting one or more non-lungworm parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria may be, for example, one or more antibodies or one or more antigens recognized by an antibody specific for one or more non-lungworm parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria.

[0109] For example, when the device of the present disclosure includes a reagent for specifically detecting hookworms, a reagent for specifically detecting lungworms, and a reagent for specifically detecting roundworms in addition to a reagent for detecting lungworms, the method of the present disclosure may involve using the device for one or more additional purposes to determine whether the sample examined for lungworms also contains hookworms, whipworms, roundworms, and / or heartworms.In this arrangement, the method / device of the present disclosure may not only be able to specifically confirm whether lungworms are present or absent in any particular test sample, but may also be useful for specifically confirming whether the sample contains or does not contain any hookworm antigens, any whipworm-derived antigens, and / or any roundworm antigens.It may be useful for the caregiver of the animal from which the test sample is obtained if a single sample can be applied to the device of the present disclosure to specifically detect lungworms and one or more other organisms. If a caregiver knows that the sample contains both lungworms and roundworms, but does not contain whipworms or hookworms, he or she can use this knowledge to treat the mammal from which the sample was taken specifically for lungworms, for example, by administering to the mammal a drug that is optimally effective against lungworms and a second drug that is optimally effective against roundworms.Without such knowledge, the caregiver may treat the mammal differently, for example, with a drug that is optimally effective only against lungworms, only against roundworms, or that is neither effective against lungworms nor against roundworms (in such cases, the mammal will be at risk of receiving a suboptimal treatment).In addition, humans who may come into contact with infested animals or their excretions may be recommended to be cautious about infestation with one or more parasites. In this context, it is generally accepted that lungworms do not play a significant zoonotic role in humans, but some parasitic helminths, such as roundworms and hookworms, can cause significant disease in humans (i.e. larva migrans, severe enteritis, or allergic reactions), so it is important to determine the helminth species with high specificity.

[0110] The method may optionally further comprise a step of determining the presence or absence of lungworms in a mammalian sample using one or more nucleic acids derived from lungworms, including but not limited to the nucleic acids of the present disclosure. Such use of these nucleic acids to determine the presence of lungworms may be performed before, after, or simultaneously with performing any other aspect of the method, including detection of lungworms by antibodies. Thus, in one aspect, after detection or non-detection of lungworms in a particular sample, the mammal from which the sample was obtained is diagnosed as having or not having lungworm infection, and the sample (or a subsequently obtained sample from the diagnosed mammal) is examined for the presence or absence of any one or more of the nucleic acids, including any one or more of the nucleic acids of the present disclosure. Any caregiver who is unable to detect lungworms in a particular mammal by using one or more nucleic acids (after detection of lungworms by using one or more antibodies) will need to take into account the possibility that the antibody detected lungworm antigens before the appearance of detectable lungworm nucleic acid in the sample. In such a case, the mammalian caregiver may choose to ignore the observation that the nucleic acid was unable to detect the lungworm, and proceed with the mammalian treatment specific for lungworm infection based on the observation that the antibody did in fact detect the lungworm.In another embodiment, the nucleic acid is used to determine the presence or absence of lungworms in a particular mammal, and then the presence or absence of lungworms is further assessed by using the antibody of the present disclosure.Detection of one or more lungworm nucleic acids may be performed by using any nucleic acid detection method known to those skilled in the art.For example, such detection may be performed by performing PCR-based techniques, such as, but not limited to, real-time PCR-based techniques.Exemplary PCR-based techniques are described, for example, in "PCR Protocols" ("Methods in Molecular Biology"), 2nd ed., Bartlett and Stirling, eds., Humana Press (2003); and Sambrook and Russell, "Molecular Cloning: Laboratory Manual", Cold Spring Harbor Laboratory Press (2001), each of which is incorporated herein by reference in its entirety.

[0111] Vaccines and pharmaceutical compositions In another aspect of the disclosure, vaccines and pharmaceutical compositions are provided for preventing A. abstrusus infection and its complications in mammals. The pharmaceutical compositions generally include one or more polypeptides or one or more polynucleotides encoding one or more of the polypeptides that contain one or more epitopes from A. abstrusus proteins, and a physiologically acceptable carrier. The vaccines include one or more of the above polypeptides or polynucleotides and an adjuvant to enhance the immune response.

[0112] The above-defined polypeptides may be used to prepare immunogenic compositions, in particular to prepare anti- A. abstrusus vaccines, but also polynucleotides encoding these polypeptides.

[0113] The immunogenic composition comprising one or more polypeptides, or one or more polynucleotides encoding said polypeptide(s), as defined above, may be combined with one or more adjuvants to enhance the immune response. In some embodiments, the composition may comprise one or more other immunogenic polypeptides recognized by anti-A.abstrusus antibodies.

[0114] In some embodiments, an immunogenic composition according to the present disclosure is a vaccine.

[0115] Where appropriate, especially when the peptide is short (≦30 amino acids), said polypeptide(s) can be coupled to a carrier protein. Examples of carrier proteins include, inter alia, KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin, tetanus toxoid or diphtheria toxoid. It is also possible to form multi-epitope compositions by combining several copies of the same peptide with each other, and optionally with other peptide epitopes, in the form of chimeric polypeptides, and also by polymer chains, such as polylysine.

[0116] When a polynucleotide is used as an immunogen, the immunogenic composition may be in the form of a recombinant vector into which the polynucleotide(s) to be administered are inserted. For example, viral vectors such as poxvirus, adenovirus, retrovirus, lentivirus, herpesvirus, and AAV (adeno-associated virus) may be used. The immunogenic composition may also be in the form of a non-pathogenic bacterium transformed with one or more expression vectors containing said polynucleotide(s). It is also possible to directly administer the polynucleotide(s) in the form of naked DNA, or to incorporate it(s) into liposomes. In the case of a vaccine, vectors derived from non-pathogenic bacteria (e.g., lactobacilli, or non-pathogenic strains of Escherichia coli or Salmonella suis), or from vaccine virus strains, may be useful.

[0117] In one embodiment, the polynucleotide of the present disclosure may be administered to a mammal in a form that allows expression of the molecule into a protective protein (e.g., mRNA) or protective RNA (e.g., antisense RNA, ribozyme, or RNA drug) in the mammal to be protected from disease. The nucleic acid molecule may be delivered to the mammal in a variety of ways, including, but not limited to, (a) direct injection (e.g., "naked" DNA or RNA molecules as taught in Wolff et al., 1990, Science, 247, 1465-1468), (b) packaging as a recombinant virus particle vaccine or recombinant cell vaccine (i.e., delivered to cells by a vehicle selected from the group consisting of recombinant virus particle vaccines and recombinant cell vaccines), or (c) encapsulation within liposomes or lipid nanoparticles. In some embodiments, the polynucleotide may include one or more modified uridine residues, such as pseudouridine or methylpseudouridine.

[0118] The recombinant virus particle vaccine of the present disclosure comprises the recombinant molecule of the present disclosure that is packaged in a viral envelope and can be expressed in an animal after administration.Preferably, the recombinant molecule is a packaging-deficient molecule.Several recombinant virus particles can be used, including but not limited to recombinant virus particles based on alphavirus, poxvirus, adenovirus, herpesvirus, and retrovirus.Methods of making and using recombinant virus particle vaccines are known in the art.

[0119] Any of a wide variety of adjuvants may be utilized in the immunogenic compositions of the present disclosure to non-specifically enhance immune responses, for example by increasing the immunogenicity of polypeptides. Most adjuvants contain substances designed to protect antigens from rapid catabolism, such as alum (aluminum hydroxide) or mineral oil, and non-specific stimulators of immune responses, such as lipid A, Bordella pertussis, or Mycobacterium tuberculosis. Such adjuvants are commercially available, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.), as well as Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), liposomes, virosomes (reconstituted viral envelopes), peptide derivatives of muramic acid, and the like. In the case of a vaccine, of course, a pharmacologically acceptable adjuvant will be chosen; by way of example of an adjuvant, reference will be made to adjuvants of the "oil-in-water" emulsion type.

[0120] In the pharmaceutical composition of the present disclosure, any suitable carrier known to those skilled in the art can be used, but the type of carrier will vary according to the mode of administration.For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer.For oral administration, any of the above carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate.Biodegradable microspheres (e.g., polylactic galactide) can also be used as carriers for the pharmaceutical composition of the present disclosure.

[0121] The route and frequency of administration, as well as the polypeptide or polynucleotide dose, may vary between individuals and may be similar to those routes and frequencies and doses currently used in immunization against other protozoan infections. In general, pharmaceutical compositions and vaccines may be administered by injection (e.g., intramuscular, intravenous, or subcutaneous), intranasally (e.g., by aspiration), or orally. Between one and four doses may be administered over a period of two to six weeks. In one embodiment, two doses are administered, with the second dose being administered two to four weeks after the first dose. A suitable dose is an amount of polypeptide that is effective to elicit sufficient antibodies in the treated mammal to protect the mammal against A. abstrusus infection over a period of time. In general, the amount of polypeptide present in a dose ranges from about 1 μg to about 100 mg, typically about 10 μg to about 1 mg, and preferably about 100 μg to about 1 μg per kg of host. Suitable dose sizes will vary with the size of the animal, but typically range from about 0.01 mL to about 5 mL for a 10-60 kg animal.

[0122] Although the present disclosure will be specifically described with reference to certain specific embodiments, the present disclosure should not be construed as being limited thereto.

[0123] Working Example Unless otherwise indicated, the following materials and techniques are used to generate the data described in one or more of Examples 1-12 below.

[0124] Preparation of lungworm extracts: The worm A. abstrusus was obtained from Dr. Bowman, Cornell University, Ithaca, NY. All worms were washed at room temperature with cold PBS, pH 7.0, multiple times to remove any fecal material and mucus from the host, and homogenized at 4°C with an ultrasonic homogenizer until no obvious tissue clumps were visible. The homogenized material was centrifuged at 10,000g for 30 minutes at 4°C, and the supernatant was carefully removed. Protein concentration was determined using the Bradford assay.

[0125] Preparation of heartworm extracts: Heartworms [Dirofilaria immitis] were obtained from IDEXX Laboratories, Westbrook, ME. All worms were disrupted, suspended in cold PBS, pH 7.0, and homogenized with a Polytron homogenizer at 4°C. The homogenized material was centrifuged at 10,000g for 30 min at 4°C, and the supernatant was carefully removed and used directly in experiment 3. Protein concentrations were determined using the Bradford assay.

[0126] Preparation of roundworm extract: Roundworms [Toxocara spp.] were obtained from Antibody systems Inc, Hurst, TX, and processed as described in U.S. Patent No. 7,951,547, which is incorporated by reference in its entirety. All worms were disrupted, suspended in cold PBS, pH 7.0, and homogenized with a Polytron homogenizer (Kinematica, Bohemia, NY) at 4° C. The homogenized material was centrifuged at 10,000 g for 30 minutes at 4° C., and the supernatant was carefully removed and used directly in experiment 4. Protein concentration was determined using the Bradford assay.

[0127] Larval excretion procedure: To detect feline lungworm, a visual fecal Baermann test is typically utilized to detect larvae, but not eggs, of the parasite in the feces (see the Baermann method described in AJ Carruth et al., Journal of Feline Medicine and Surgery, 2019, Vol. 5, pp. 1-6).

[0128] Preparation of feline fecal samples: felines that are not infected with lungworm or are known to be infected with lungworm provide the source of fecal samples. Samples (approximately 1 gram) from frozen, unpreserved fecal samples were suspended in 4 ml of diluent ("diluent" is 0.05M Tris base; 1 mM EDTA; 0.45% Kathon; 16 mg / l gentamicin sulfate; 0.05% Tween-20; 40% fetal bovine serum; 10% rabbit serum; and 5% mouse serum). The suspension was centrifuged in a tabletop centrifuge at 4000 rpm for 20 minutes to obtain a first supernatant. The first supernatant was centrifuged at 10,000 g for 5 minutes to obtain a second supernatant, which is referred to herein as "fecal extract".

[0129] Preparation of feline serum samples: Felines known to be either lungworm-free or lungworm-infected provided the source of serum samples. Serum samples from six experimentally infected cats were obtained from Dr. Bowman's laboratory at Cornell University and were used throughout this study. Cats were infected at approximately 12-14 weeks of age. Each cat received 250 third stage larvae (L3) taken from a laboratory colony of the aquatic snail Biomphalaria glabrata to initiate their infection. Weekly fecal samples were collected and the Baermann procedure was performed to calculate the larval shedding rate in terms of larvae per gram for each cat. Weekly blood draws were also performed with samples taken from alternating medial saphenous veins. Experimental infection continued until day 89, at which point imidacloprid and moxidectin therapy (Advantage Multi, Bayer) was introduced. Cats received at least two doses of this regimen over a two month period, with weekly stool and blood sampling performed up to day 189.

[0130] Preparation of recombinant TDX1557 protein: A codon-optimized version of the 2M2 gene (SEQ ID NO: 19) was synthesized and cloned into pCDNA 3.1 (TDX1557).

[0131] To purify recombinant TDX1557, sequences encoding a HIS tag and an ADX18 epitope tag were added to the construct (SEQ ID NO: 20) at the C-terminal region (see FIG. 1, part B).

[0132] In preparing the construct (SEQ ID NO: 20), a sequence encoding an IgG-like signal sequence was added so that the recombinant 2M2 (rTDX1557) polypeptide (SEQ ID NO: 3) was secreted into the supernatant. MDWTWRVFFLLALATGVHSEN (SEQ ID NO:21) was incorporated into the amino terminal region of 2M2, at positions 941-1003 of SEQ ID NO:2. [ka] The recombinant protein was tagged with β-lactamase to facilitate purification. The recombinant construct was transfected into a mammalian expression system (HEK293 cells) using the ExpiFectamine™ Transfection Kit (Thermo Fisher Scientific, Carlsbad, CA). The supernatant was harvested after 5-7 days and the recombinant protein TCX1557 (SEQ ID NO:3) was purified using immobilized metal affinity chromatography (IMAC). [ka]

[0133] Preparation of polyclonal lungworm antibodies: In each rabbit, a polyclonal antibody "anti-TDX1557 pAB" (IgG) was raised against a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 3, and purified from serum by using standard methods. After introducing rTDX1557 into the rabbit, anti-TDX1557 pAB was purified from the plasma of the immunized rabbit by isolating IgG antibodies via protein G affinity chromatography.

[0134] Infection of felines: Infection with the parasitic lungworm nematode was performed by oral ingestion of approximately 150-300 [infective larvae of A. abstrusus] to healthy cats. Infection was confirmed by microscopic observation of helminth eggs in fecal samples obtained from these host animals. Larval excretion was performed using the standard Baermann technique (AJ Carruth et al., Journal of Feline Medicine and Surgery, 2019, Vol. 5, pp. 1-6). Infected cats were treated with the antiparasitic drugs imidacloprid and moxidectin on day 90.

[0135] ELISA assay: Lung nematode lysate or recombinant TDX1557 ("rTDX1557") (100μl per well; 3μg / ml for rTDX1557 and approximately 2ug / mL for lysate) was immobilized on Immulon IV 96-well plate by physical adsorption overnight at 4°C. Plate was then blocked with 1% BSA in 0.1M Tris pH 7.0 for 2 hours at room temperature. Well was then washed 5 times with PBS-Tween-20 solution according to standard methods known to those skilled in the art. Serum samples from experimentally infected cats were diluted with PRRS diluent (IDEXX PRRS X3 Ab Test, model number: 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) at a dilution ratio of 1:200 (100μl per well) and added to well. After incubation at room temperature for 1 hour, the wells were washed five times using PBS-Tween-20 solution according to standard methods known to those skilled in the art. Then, 50 μl of goat anti-cat IgG(H+L)-HRP antibody (Jackson ImmunoResearch Inc., West Grove, PA) diluted 1:5000 in Enzyme conjugate diluent (IDEXX Laboratories, Westbrook, ME) was added to each well, and the plate was incubated at room temperature for 30 minutes. The plate was washed, and 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (IDEXX Laboratories, Westbrook, ME) was added to each well, and the plate was incubated at room temperature for 15 minutes. After stopping each enzymatic reaction with malic acid, the optical density (OD) value of each well of the 96-well plate was measured at A450 by using an ELISA plate reader that provided an "OD450 value" (or more simply, "OD value") for each well via standard spectrophotometric methods. With this setup, the OD value obtained for any particular well of the 96-well plate was directly proportional to the amount of specific bound antibody present in the well.

[0136] Immunoblot procedure: Worm lysates (3ug / ml) or recombinant TDX1557 (10ug) were mixed with SDS-PAGE sample buffer and subjected to SDS-PAGE gel electrophoresis at 200V for 35 minutes (Thermo Fisher, Carlsbad, MA). Total protein lysates from SDS-PAGE gels were transferred to nitrocellulose (NC) membranes using iblot (Thermo Fisher, Carlsbad, CA). The NC membranes were probed with rabbit polyclonal antibody against TDX1557 followed by the addition of alkaline phosphatase-coupled anti-rabbit IgG H+L (Sigma, St Louis, MO). Blots were developed with BCIP / NBT substrate (Sigma, St Louis, MO).

[0137] ELISA procedure using Neutravidin plates for peptide array analysis: Neutravidin plates (Thermo Fisher, Carlsbad, CA) were coated with 100ul of PBS-diluted peptides in the Neutravidin plates, and the plates were incubated at room temperature for 1 hour with shaking. The plates were then washed 3 times with PBS-T. Cat serum samples were diluted 1:800 in 1x PBS (pH 7.2 + 2% BSA), 100ul (unknown concentration) was applied per well, and the plates were incubated at room temperature for 1 hour with shaking. The plates were then washed 3 times with PBS-T. 100ul of secondary antibody dilution was added to each well, followed by 12ul of goat anti-cat H+L HRP in 60mls ECD. The plates were then incubated at room temperature for 1 hour with shaking, washed 5 times with PBS-T, and then developed with 100ul of TMB substrate. Stop solution was added and the solution was read at 450 nm.

[0138] Example 1 Preparation and characterization of recombinant TDX1557 In this example, recombinant TDX1557 (also referred to as recombinant 2M2 or rTDX1557) (SEQ ID NO: 3) was expressed and purified in a mammalian transfected HEK293 cell line as discussed above. The purified protein was analyzed by SDS-PAGE electrophoresis. The predicted molecular weight of 2M2 (TDX1557) is 28 kDa, while the molecular weight of recombinant TDX1557 was 62 KDa due to post-translational modification in the mammalian expression system. To further confirm that recombinant TDX protein was produced, immunoblot was performed as described by the procedure above. The gel was blotted onto a nitrocellulose membrane and probed with an alkaline phosphatase tagged monoclonal antibody against the HIS tag (Sigma, St Louis, MO). Substrate NBT / BCIP (Sigma, St Louis, MO) was added for development of the blot as described by the procedure above. The gel and immunoblot results are shown in Figure 1.

[0139] SDS-PAGE analysis of the supernatant from the mammalian expression system confirmed the expression of recombinant 2M2, as shown in Figure 1, part A. Immunoblot analysis of recombinant 2M2 is shown in Figure 1, part B. Recombinant 2M2 was probed with mAb HIS, tagged with alkaline phosphatase, as described above, followed by development with BCIP / NBT.

[0140] Example 2 Detection of lungworm infection using an ELISA assay with TDX1557 This example compares detection and measurement of lungworm infection using a fecal Baermann assay with detection and measurement of lungworm infection using an indirect ELISA, using lungworm lysates and rTDX1557.

[0141] Experimental infection of cats and procedures: Cats were orally infected with A. abstrusus larvae as described above. Fecal samples and blood samples were collected over a defined time period (days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 112, 126, 140, 154, 168, and 182). Infected cats were treated with the antiparasitic drugs imidacloprid and moxidectin by day 90. The Behrmann method for visual detection and counting of lungworm larvae was performed on fecal samples using standard procedures (AJ Carruth et al., JFMS, 2019, vol. 5, pp. 1-6). Serum samples collected over a defined time period were assessed in an indirect ELISA assay with lungworm lysates and rTDX1557. A. abstrusus lysates were prepared from lungworm larvae as described above. Total protein in the lysates was determined by conventional Bradford assay. Recombinant protein TDX1557 was obtained as described above.

[0142] ELISA assay for Aelurostrongylus abstrusus lysate and rTDX1557 The lungworm larval excretion rate was determined by the standard Behrmann method using fecal samples from experimentally infected samples. Using the above procedure, lungworm lysates and indirect ELISA assays for rTDX1557 were performed. Two cats (BigMac and Burrito) were experimentally infected with lungworms, and serum samples from these cats were diluted in PRRS buffer (IDEXX PRRS X3 Ab Test, model number: 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) and used for the ELISA procedure.

[0143] Immulon IV plates were coated with lungworm lysate or rTDX1557 overnight at 4° C. and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5×PBST and serum samples from experimentally infected cats (BigMAC or Burrito) were diluted in PRRS diluent and added to the plates and held at room temperature for 1 hour. The plates were washed again with 5×PBST and secondary antibody (anti-cat IgG H+L) was added to the plates and held for 30 minutes. The plates were washed, substrate was added and read at 450 nm.

[0144] Larval shedding results for experimentally infected cats are shown in Figure 2, parts A and B, where larval shedding rates were measured over a period of 182 days. ELISA assays were performed with A. abstrusus lysate and rTDX1557(2M2) to compare larval shedding rates in high shedding cats ("BigMac") (see Figure 2, part A) with larval shedding rates in low shedding cats ("Burrito") (see Figure 2, part B). The X-axis indicates days post-infection (DPI). The Y1 and Y2 axes represent OD values, which reflect shedding rates, respectively. The results indicate that larval shedding measured by fecal Baermann method resulted in inconsistencies, but ELISA assays with lungworm lysate and rTDX1557 showed a gradual increase in antibody titers against lungworm, while the treated cats responded to treatment, as antibody titers dropped after day 90. The ELISA assay with recombinant TDX1557 shows a much more robust antibody response compared to the ELISA assay with lungworm lysate.

[0145] Example 3 Absence of cross-reactivity of anti-TDX1557 with Dirofilaria immitis This example illustrates that rabbit polyclonal antibodies raised against rTDX1557 did not cross-react with Dirofilaria immitis using SDS-PAGE and immunoblot analysis.

[0146] SDS-PAGE and immunoblot analysis of A. abstrusus and Dirofilaria immitis lysates Rabbit polyclonal antibodies against rTDX1557, D. immitis lysate, and D. pulmonis lysate were prepared as described above. rTDX1557, D. immitis lysate, and D. pulmonis lysate were mixed with SDS-PAGE buffer separately and subjected to SDS-PAGE gel electrophoresis in two different gels using conventional procedures. In the first gel, rTDX1557 (lane 1), A. abstrusus lysate (lane 3), and D. immitis lysate (lane 4) were stained with Imperial stain. The second gel with the same lane was subjected to immunoblot analysis by transferring the total protein of lysate to nitrocellulose (NC) membrane. The NC membrane was probed with rabbit polyclonal antibody against TDX1557 followed by addition of alkaline phosphatase conjugate (alkaline phosphatase coupled anti-rabbit IgG H+L) (Sigma, St Louis, MO). Blots were developed with BCIP / NBT substrate. The positive control was rTDX1557, with a molecular weight of approximately 62 kDA. Lane 2 was left blank. The results are shown in Figure 3, parts A and B.

[0147] As shown in Figure 3, part A, SDA-PAGE analysis of A. abstrusus lysate and D. immitis lysate shows expression of 2M2 (TDX1557) in the pulmonary nematode, C. immitis. Figure 3, part B shows immunoblot analysis of A. abstrusus lysate and D. immitis lysate after probing with rabbit polyclonal antibody against TDX1557 (anti-TDX1557). The rabbit polyclonal antibody against TDX1557 recognized a single band of approximately 62 kDa in the A. abstrusus lysate but did not cross-react with D. immitis lysate.

[0148] Example 4 Absence of anti-TDX1557 cross-reactivity with Ascaris This example illustrates that rabbit polyclonal antibodies raised against rTDX1557 did not cross-react with roundworms (Toxocara spp.) using SDS-PAGE and immunoblot analysis.

[0149] SDS-PAGE and immunoblot analysis of A. abstrusus and Ascaris lysates Rabbit polyclonal antibodies against rTDX1557, roundworm antigen (Toxocara spp.), and lungworm antigen were prepared as described above. In the SDS-PAGE gel, rTDX1557 (lane B), lungworm A. abstrusus lysate (lane C), and lungworm lysate (lane D) were mixed with SDS-PAGE buffer separately and subjected to SDS-PAGE gel electrophoresis using routine procedures. The resulting gel was subjected to immunoblot analysis by transferring the total proteins in the gel to NC membrane. The NC membrane was probed with rabbit polyclonal antibody against TDX1557, followed by addition of alkaline phosphatase conjugate (alkaline phosphatase-coupled anti-rabbit IgG H+L, Sigma, St. Louis, MO). The blot was developed with BCIP / NBT substrate (Sigma, St. Louis, MO). The positive control was rTDX1557. The results are shown in Figure 4.

[0150] Figure 4 shows immunoblot analysis of A. abstrusus lysates and Ascaris lysates probed with rabbit polyclonal antibody against TDX1557 (anti-TDX1557) prepared as described above. The rabbit polyclonal antibody against TDX1557 recognized a single band pellet of approximately 62 kDa in A. abstrusus lysates (lane C) but did not cross-react with Ascaris soluble antigens (lane D).

[0151] Example 5 Detection of Pulmonary Nematode Infection Using ELISA and the SNAP® Test with TDX1557 This example compares detection and measurement of lungworm infection using a fecal Baermann assay (results from Example 2) and an indirect ELISA assay (results from Example 2) to detection and measurement of lungworm infection using the SNAP diagnostic device using lungworm lysate and rTDX1557 as a positive control.

[0152] Experimental cat infection: Cats (BigMac) were orally infected with A.abstrusus larvae as described in Example 2. Fecal samples and blood samples were collected over a defined period (days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 112, 126, 140, 154, 168, and 182). Infected cats were treated with the antiparasitic drugs imidacloprid and moxidectin by day 90. The Behrmann method for visual detection and counting lungworm larvae was performed on fecal samples using standard procedures (AJ Carruth et al., Journal of Feline Medicine and Surgery, 2019, vol. 5, pp. 1-6). Serum samples collected during the specified time period were assessed in an indirect ELISA assay with lungworm lysate and rTDX1557 as a control, and in a SNAP assay device using rTDX1557 as a control. A.abstrusus lysate was prepared from lungworm larvae as described above. Total protein in the lysate was determined by conventional Bradford assay. Recombinant protein TDX1557 was obtained as described above.

[0153] ELISA assay for A. abstrusus lysates and rTDX1557 Using the above procedure, lungworm lysates and indirect ELISA assays for rTDX1557 were performed. Two cats (BigMac and Burrito) were experimentally infected with lungworms, and serum samples from these cats were diluted in PRRS buffer and used for the ELISA procedure.

[0154] Immulon IV plates were coated with lungworm lysate or rTDX1557 overnight at 4° C., and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5×PBST, and serum samples from experimentally infected cats (BigMAC) were diluted in PRRS diluent and added to the plates and held at room temperature for 1 hour. The plates were washed again with 5×PBST, and secondary antibody (anti-cat IgG H+L) was added to the plates and held for 30 minutes. The plates were washed, substrate was added, and read at 450 nm. The results of the indirect ELISA assay using lungworm lysate and rTDX1557 as controls are shown in FIG. 5A (replicated from FIG. 2A).

[0155] SNAP assay Recombinant TDX1557 was passively coated onto polystyrene particles (Sphero, Lake Forest, IL) at 1 mg / mL in PBS (1% solids) and 0.7 ul was spotted onto the matrix (IDEXX Laboratories). The positive control was goat anti-HRPO coated latex particles. SNAP devices were assembled with wash buffer (IDEXX Laboratories). Recombinant TDX1557 was also covalently linked to HRPO by periodate conjugation. The conjugate (180 ul) was mixed with 150 ul of serum sample and added to the sample well. Once the sample reached the activation circle, the SNAP device was activated and washed with matrix followed by development with substrate. SNAP results were read visually by the naked eye and by densitometer. SNAP results are shown in Figure 5, part C. The results of larval excretion and indirect ELISA assay are summarized in Figure 5, part A (replicated from Figure 2, part A). As shown in Figure 5, part A, comparison of fecal results using the standard Baermann test showed inconsistency in larval shedding compared to indirect ELISA results using serum samples, which showed a stable evolution of antibody responses to rTDX1557 and A. abstrusus lysates over time.

[0156] The SNAP results using serum samples are summarized in Figure 5, part C. Antibody reactivity was observed from day 14 onwards.

[0157] A numerical comparison of the Baermann test (larval shedding), indirect ELISA (with rTDX1557), and SNAP results is shown in Figure 5, part B. Compared to the larval shedding and indirect ELISA assays, the SNAP assay results should detect lungworm infection as early as day 14 after experimental infection. In contrast, the indirect ELISA and larval tests showed detection of lungworm infection from experimental infection on days 35 and 63. The SNAP results for the 10 cat field samples show good correlation with the negative indirect ELISA, except for one case.

[0158] Example 6 Construction, expression, and characterization of truncated forms of TDX1557 This example describes the construction of truncated forms of 2M2 / TDX1557 and the assessment of these truncated 2M2 peptides in an indirect ELISA assay using rabbit pAbs against 2M2, time-course samples (Burrito DPI 98 and French Fry DPI 91), and negative samples (SPF 16CMF3, SPF 16CSL3). The amino acid sequences of the four truncations are shown in Figure 6. The truncated forms of TDX1557 labeled as T2 (also referred to as 2M2_T2 or TDX1637), T3 (also referred to as 2M2_T3 or TDX1674), and T4 (also referred to as 2M2_T4 or TDX1675) were cloned and expressed as discussed above for rTDX1557.

[0159] Construct for T2 (SEQ ID NO:23) ATGGACTGGACTTGGAGAGTGTTCTTCCTGCTGGCCCTGGCTACAGGCGTGCACAGCGAGAACATGAACGACCAGCTGCGGGACATCATGCTGAACGATCACAACACCCTGAGAAGCCTGGCCGCCAAAGGACTGGCCGAAAATCCTCTGGGCACCAACGGCAGAGCCCCTAAAGCTGCCAGAATGCTGAAGATGGTGTACGACTGCAACGTGGAAAAGACCGCCATGATCCACGCCAAGAAATGCGTGTTCGAGCACAGCAAGGGCAGAAAGGACACCGGCGAGAACGTGTGGGTTATGTGGCCCGCTCAGAAAAACCTGACCGAGATGGCCACCAGAGCCAGCAAGAGTTGGTTCGACGAGCTGAAGAAATTCGGCGTGCCACCTGACAACATCCTGACCGAAGGCCTGTGGAACAGACCCAAGATGGCCATTGGCCACTACACCCAGATGGTCTGGGAGGGCTCTTACAAGCTCGGATGTGGCGTGGCCACATGCAGCGACAAGACCCTGATCGTGTGCCAGTATAGCCCTGCCGGCAACTACATCGGCAGCATCATCTATGCCATCGGCGAGCCCTGCAAGACCGACGAGGATTGCAAGTGCGAGGGCTGCAAGTGCAGCAGAGATGAGGCCCTGTGCATCAAGCCTAATGCCGCCGCTCACCACCATCATCACCATCACCACGGTGTTCTGGCCCCTCACGATTCTGTTCTGCAGTAATAA

[0160] Construct for T3 (SEQ ID NO: 24) ATGGACTGGACTTGGAGAGTGTTCTTCCTGCTGGCCCTGGCTACAGGCGTGCACAGCGAGAACAACGACGAGATGGACGAGAACAATCCCGTGGCTCTGCCCGGCAAGAGCAATGTGTCTGAGCCTTGCCAGACCACCACCAGCAATCCTATTCACCCTACCACCGGCGAGCAGCTGGAAACCACAACAGGCAAGCCCAGACGGACCACCAAGGGCAGAATCCTGAAAACCACCACCGTGAAGCCCATCAAGACAACAAAGGGCAAGAAGCTCAAGACGACCAAGGGGAAGCTGCTGAAAACAACAACAGTCAAACCCTTCAAGACCACGAAGGGAAAGCCCCTTAAGACAACCACCAAAGAGCCCTTCCAGAGCACCACAGGTGGACCAGTGGGCCCAACCACCGATAGATGCAAGCTGAACAACGGCATGAACGACCAGCTGCGGGACATCATGCTGAACGATCACAACACCCTGAGAAGCCTGGCCGCCAAAGGACTGGCCGAAAATCCTCTGGGCACCAACGGCAGAGCCCCTAAAGCTGCCAGAATGCTGAAGATGGTGTACGACTGCAACGTGGAAAAGACCGCCATGATCCACGCCAAGAAATGCGTGTTCGAGCACAGCAAAGGCCGGAAGGATACAGGCGAAGCCGCCGCTCACCACCATCATCACCATCACCACGGTGTTCTGGCCCCTCACGATTCTGTTCTGCAGTAATAA

[0161] Construct for T4 (SEQ ID NO: 25) ATGGACTGGACTTGGAGAGTGTTCTTCCTGCTGGCCCTGGCTACAGGCGTGCACTCTGAGAACGGCAGAAAGGACACCGGCGAGAACGTGTGGGTTATGTGGCCCGCTCAGAAAAACCTGACCGAGATGGC CACCAGAGCCAGCAAGAGTTGGTTCGACGAGCTGAAGAAATTCGGCGTGCCACCTGACAACATCCTGACCGAAGGCCTGTGGAACAGACCCAAGATGGCCATTGGCCACTACACCCAGATGGTCTGGGAGG GCTCTTACAAGCTCGGATGTGGCGTGGCCACATGCAGCGACAAGACCCTGATCGTGTGCCAGTATAGCCCTGCCGGCAACTACATCGGCAGCATCATCTATGCCATCGGCGAGCCCTGCAAGACCGACGAG GATTGCAAGTGCGAGGGCTGCAAGTGCAGCAGAGATGAGGCCCTGTGCATCAAGCCTAATGCCGCCGCTCACCACCATCATCACCATCACCACGGTGTTCTGGCCCCTCACGATTCTGTTCTGCAGTAATAA

[0162] In making the construct, the truncated form was added with an IgG-like signal sequence, [ka] was incorporated into the amino-terminal region of each truncated form at positions 1-63 of SEQ ID NO:23 (T2 construct), at positions 1-63 of SEQ ID NO:24 (T3 construct), and at positions 1-63 of SEQ ID NO:25 (T4 construct). [ka] The recombinant truncated proteins were tagged with β-glucose to facilitate purification. The recombinant constructs were transfected into a mammalian expression system (HEK293 cells) using the ExpiFectamine™ Transfection Kit (Thermo Fisher Scientific, Carlsbad, CA). Supernatants were collected after 5-7 days, and the recombinant truncated proteins were purified using immobilized metal affinity chromatography (IMAC). The predicted structures [https: / / wlab.ethz.ch / protter / ] indicate secondary structures such as helical structures and beta sheets of the truncated forms. SDS-PAGE analysis shows the expression of each truncated TDX1557 protein. 2M2(TDX1557). MDWTWRVFFLLALATGVHSEN NDEMDENNPVALPGKSNVSEPCQTTTSNPIHPTTGEQLETTTGKPRRTTKGRILKTTTVKPIKTTKGKKLKTTKGKLLKTTTVKPFKTTKGKPLKTTTKEPFQSTTGGPVGPTTDRCKLNNGMNDQLRDIMLNDHNTLRSLAAKGLAENPLGTNGRAPKAARMLKMVYDCNVEKTAMIHAKKCVFEHSKGRKDTGENVWVMWPAQKNLTEMATRASKSWFDELKKFGVPPDNILTEGLWNRPKMAIGHYTQMVWEGSYKLGCGVATCSDKTLIVCQYSPAGNYIGSIIYAIGEPCKTDEDCKCEGCKCSRDEALCIKPNHHHHHHHGVLAPHDSVLQ (SEQ ID NO: 3) [ka]

[0163] The indirect ELISA procedure used in this example was as described above. Briefly, separate Immulon IV plates were coated with rTDX1557 and each of the recombinant truncations, T2, T3, and T4, overnight at 4° C., and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5×PBST, and serum samples from experimentally infected cats were diluted in PRRS diluent and added to the plates, and the plates were held at room temperature for 1 hour. The plates were washed again with 5×PBST, and secondary antibody (anti-cat IgG H+L) was added to the plates, and the plates were held for 30 minutes. The plates were washed, substrate was added, and read at 450 nm. The results are shown in FIG. 8.

[0164] Figure 8 shows the assessment of truncated forms of 2M2 (TDX1557) using an indirect ELISA assay. Four different variations of recombinant peptides (2M2 full length, T2, T3, and T4) were assessed in an indirect ELISA assay using rabbit pAb, time-course samples (Burrito DPI 98, French Fry DPI 91), and negative samples (SPF 16CMF3, SPF 16CSL3). Serially diluted serum samples were examined at different concentrations of recombinant peptides (0.5, 1.5, and 3ug / ml). Rabbit polyclonal antibodies against TDX1557 reacted with all four different recombinant peptides. However, experimental infection samples did not show any reactivity with the 2M2 T3 truncations, indicating that a prominent epitope exists only at the carboxy terminus of the protein. The specific pathogen free (SPF) negative controls (SPF cat ID: 16CMF3 and 16CSL3) show no reactivity to any of the recombinant peptides. Based on antigen concentration and dilution, an increased titer rate was also observed, indicating an antibody response to the truncated forms.

[0165] The truncation epitopes 2M2 Amino2 Cys, 2M2 CpepC, 2M2 Wo, and Cys-Ser truncation mutants of 2M2 T4 (referred to as 2M2 T4 WO IDR, 2M2 T4 1A, 2M2 T4 1B, and 2M2 T4 1C) were also constructed. See Figure 16C1. The sequences for these truncations are shown in Figure 16C2. The truncations 2M2 Amino2 Cys, 2M2 CpepC were successfully expressed, but the Cys-Ser truncation mutants were not. Figures 16D and 16E show the assessment of the epitopes 2M2 Amino2 Cys, 2M2 CpepC, 2M2 Wo using the above procedure. As shown in Figures 16D and 16E, no reactivity was found for the time-lapse samples.

[0166] Example 7A Identification of epitopes of TDX1557 using peptide arrays This example shows the assessment of a peptide array with serum samples from uninfected cats (TDX1557 ELISA negative) based on the ELISA protocol on Neutravidin plates described above. The array incorporated 62 peptides, 15mers each, with 10 amino acid overlaps, covering the entire length of the 2M2 protein.

[0167] [Table 1-1] [Table 1-2]

[0168] These peptides were synthesized using standard procedures and labeled with biotin at the N-terminus (New England peptide, MA). Neutravidin plates (Thermofisher, Carlsbad, CA) were coated with the biotinylated peptides. Following incubation of the plates with serum samples, they were washed with PBST. Secondary antibody (anti-cat IgG H+L) with HRP conjugate was added and held at room temperature for 30 minutes. Plates were washed with PBST and substrate was added. Stop solution was added and the solution was read at 450 nm.

[0169] As shown in Figure 9, negative samples (AC03772, AC04400, and AN06876) and SPF pooled samples were used. Raw data was plotted between negative and positive samples to directly show the differences. As for experimental or time-course samples, the OD value at day 0 (pre-bleed) was used to normalize the data for this particular cat. The negative samples do not show significant reactivity to peptides compared to the positive field samples in Figure 10 (AC03791, AC03796, AC12570, AC12692, AC12693, AN00997, AN24140, and BB62456) or the experimentally infected samples in Figure 11 (BigMac 91dpi, Burrito 85dpi, Nugget 85dpi, Taco dpi85, and Tortilla dpi85).

[0170] Example 7B Identification of epitopes of TDX1557 using peptide arrays This example shows the evaluation of the peptide array of Example 7A with serum samples from naturally infected cats (TDX1557 ELISA positive) using the ELISA protocol on Neutravidin plates described above. The protein array was evaluated using serum samples collected from naturally infected cats (positive in the ELISA assay with the whole protein). The results show significant reactivity to the peptides.

[0171] Using the procedure described above, Neutravidin plates were coated with the N-terminally biotinylated peptide of Example 7A. Following incubation of the plates with serum samples, they were washed with PBST. Secondary antibody (anti-cat IgG H+L) with HRP conjugate was added and held at room temperature for 30 minutes. The plates were washed with PBST and substrate was added. Stop solution was added and the solution was read at 450 nm. The results are shown in FIG. 10.

[0172] As shown in Figure 10, the positive feline serum samples showed significant reactivity to the following peptide sequences, which were found to be the most immunogenic peptides in TDX1557:

[0173] Peptide number Peptide sequence C10 MVYDCNVEKTAMIHA (SEQ ID NO: 7) C11 NVEKTAMIHAKKCVF (SEQ ID NO: 8) C12 AMIHAKKCVFEHSKG (SEQ ID NO: 9) D01 KKCVFEHSKGRKDTG (SEQ ID NO: 10) D02 EHSKGRKDTGENVWV (SEQ ID NO: 11) D03 RKDTGENVWVMWPAQ (SEQ ID NO: 12) D04 ENVWVMWPAQKNLTE (SEQ ID NO: 13) D05 MWPAQKNLTEMATRA (SEQ ID NO: 14) D06 KNLTEMATRASKSWF (SEQ ID NO: 15) D07 MATRASKSWFDELKK (SEQ ID NO: 16) D08 SKSWFDELKKFGVPP (SEQ ID NO: 17)

[0174] Example 7C Identification of immunodominant epitopes of TDX1557 using peptide arrays This example demonstrates the assessment of the peptide array of Example 7A with serum samples taken from experimentally infected cats (TDX1557 ELISA positive) using the ELISA protocol on Neutravidin plates described above.

[0175] Using the procedure described above, Neutravidin plates (Thermofisher, Carlsbad, CA) were coated with the N-terminally biotinylated peptide of Example 7A. Following incubation of the plates with serum samples, they were washed with PBST. Secondary antibody (anti-cat IgG H+L) with HRP conjugate was added and held at room temperature for 30 minutes. The plates were washed with PBST and substrate was added. Stop solution was added and the solution was read at 450 nm. The results are shown in FIG. 11.

[0176] As shown in Figure 11, the 2M2 peptide array was assessed with eight positive experimentally infected samples (Big Mac 91 dpi, Burrito 85 dpi, Nugget 85 dpi, Taco 85 dpi, Tortilla 85 dpi) and a specific pathogen free (SPF) cat pool (negative control). Experimentally infected samples showing significant positive reactivity to the intact protein in the ELISA assay also showed significant reactivity to the peptides. The immunogenic peptide sequences of Example 7B (D6, D7, D8, C10, C11, C12, D2, D3, and D4) were found to be the most immunogenic.

[0177] Figure 11 also includes an alignment of D6, D7, and D8, where the underline indicates the overlap region shared by the three peptides with the sequence SKSWF (SEQ ID NO: 81) as the sequence that contains the epitope. The overlap region between D6 and D7 indicates that the sequence MATRASKSWF (SEQ ID NO: 82) contains the epitope. The overlap region between D7 and D8 indicates that the sequence SKSWFDELKK (SEQ ID NO: 83) contains the epitope.

[0178] Figure 11 further includes an alignment of C10, C11, and C12, where the underline indicates the overlap region shared by the three peptides with sequence AMIHA (SEQ ID NO: 84) as the sequence containing the epitope. The overlap region between C10 and C11 indicates that sequence NVEKTAMIHA (SEQ ID NO: 85) contains the epitope. The overlap region between C11 and C12 indicates that sequence AMIHAKKCVF (SEQ ID NO: 86) contains the epitope.

[0179] Figure 11 additionally includes an alignment of D2, D3, and D4, in which the overlapping region shared by the three peptides, with sequence ENVWV (SEQ ID NO: 87), contains the epitope. The overlapping region between D2 and D3 indicates that sequence RKDTGENVWV (SEQ ID NO: 88) contains the epitope. The overlapping region between D3 and D4 indicates that sequence ENVWVMWPAQ (SEQ ID NO: 89) contains the epitope.

[0180] Example 8 Assessment of Polypeptides D6, D7, and D8 This example shows a comparison of the reactivity of peptides D6, D7, and D8, A. abstrusus lysate, and rTDX1557 with antisera from experimentally infected cats. Peptides (D6, D7, D8) were prepared as described in Example 7A. Indirect ELISA assays were used to measure antibody binding to peptides D6, D7, and D8, A. abstrusus lysate, and rTDX1557. Linear peptides (D6, D7, D8) bound to antibodies at early time points indicating their diagnostic utility for detection and diagnosis of infection, including early in infection. ELISA assays with larvae lysate and rTDX1557 showed a gradual increase over time, consistent with a gradual increase in antibody titers. Antibody titers dropped after treatment with antiparasitic drugs and occurred 90 days after infection. The table below shows the OD values.

[0181] [Table 2]

[0182] FIG. 12 shows a comparison of ELISA assays with peptides (D6, D7, and D8), recombinant TDX1557, and lysates of the lung nematode A. abstrusus using serum samples from an experimentally infected high shedding cat, Tortilla.

[0183] Example 9 Assessment of the polypeptide D678 in infected cats This example describes the construction of a polypeptide, D678, and the assessment of its reactivity with serum samples taken from experimentally infected cats.

[0184] Figure 13 shows the polypeptide structure created using Protter (https: / / wlab.ethz.ch / protter / ), an open source tool used to visualize protein morphology for the construction of 2M2. Protter indicated that 2M2 is a secreted protein and contains the underlined regions of immunogenic peptides D6, D7, and D8 within the 2M2 sequence. Alignment of D06, D07, and D08 shows the overlapping SKSWF sequence (SEQ ID NO: 81). Combinatorial peptide D678 ("D678 combo") based on a combination of peptides D6, D7, and D8 was synthesized using standard peptide synthesis procedures.

[0185] D678 combo: CPAQKNLTEMATRASKSWFDELKKFG (SEQ ID NO: 18)

[0186] The N-terminal C residue in SEQ ID NO: 18 is not part of the native sequence. It was added to facilitate manipulation, e.g., conjugation, as is well known in the art. "Modified D678", SEQ ID NO: 78 below, is the same as SEQ ID NO: 18, except that it does not have the N-terminal C.

[0187] Immulon IV plates were individually coated with A. abstrusus lysate prepared as above, rTDX1557 prepared as above, and non-biotinylated synthetic peptide D678 at 4°C overnight, and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5x PBST, and serum samples from experimentally infected cats were diluted in PRRS diluent and added to the plates and incubated at room temperature for 1 hour. The plates were washed again with 5x PBST, and secondary antibody (anti-cat IgG H+L) was added to the plates and incubated at room temperature for 30 minutes. The plates were washed, substrate was added, and the plates were read at 450 nm. The results of the indirect ELISA assay using A. abstrusus lysate and rTDX1557 as controls, and peptide D678 are shown in Figure 14.

[0188] Although peptide D678 is a combination of immunogenic peptides D6, D7, and D8 that generate early antibody responses as discussed and shown in Examples 8 and 9, peptide D678 was found to be unequal in terms of its antibody response compared to the individual peptides (D6, D7, and D8). The lack of high antibody response could be due to the non-biotinylated form of D678, assessed compared to the biotinylated peptides D6, D7, and D8 used in the experiments discussed above.

[0189] Example 10 Construction of an extended polypeptide, D678, and assessment of detection of antibodies from infected cats In this example, two extended forms of the D678 peptide were constructed in which the immunogenic region was extended at either the amino terminus (D678 Amino) or the carboxyl terminus (D678 Carboxyl) and assessed via an indirect ELISA assay to determine whether one or both of the extended D678 peptides exhibited enhanced immune reactivity compared to the modified D678 peptide. The alignment of the amino acid sequences of peptides modified D678 [SEQ ID NO: 78], D678 amino [SEQ ID NO: 79], and D678 carboxy [SEQ ID NO: 80] is shown in Figure 15. The overlapping amino acids of peptide modified D678 and the extended forms of this peptide (peptides D678 amino and D678 carboxy) are underlined. Peptides D678, D678 amino, and D678 carboxy were synthesized by New England peptide (Gardner, MA).

[0190] Modification D678: PAQKNLTEMATRASKSWFDELKKFG (SEQ ID NO:78)

[0191] D678 Amino: CVFEHSKGRKDTGENVWVMW PAQKNLTEMATRASKSWFDELKKFG (SEQ ID NO:79)

[0192] D678 Carboxy: C PAQKNLTEMATRASKSWFDELKKFG VPPDNILTEGLWNRPKMAIGHYTQ (SEQ ID NO: 80) The N-terminal C residue in SEQ ID NO:80 was added to facilitate conjugation; ie, it is not part of the native sequence.

[0193] The indirect ELISA procedure used in this example was as described above. Briefly, Immulon IV plates were coated with recombinant peptides modified D678, D678 amino, and D678 carboxy at 4°C overnight, and the plates were blocked with blocking solution (1% BSA in 100mM Tris pH 7.5). The plates were washed three times with PBST, and 100uL of serum samples from infected cats were diluted with PRRS diluent (IDEXX PRRS X3 Ab Test, model number: 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) (1:100 diluted samples) and added to the plates, and the plates were kept at room temperature for 1 hour. The plate was washed once with PBS-Tween buffer, aspirated, and 100 uL of conjugate (anti-cat IGG H+L; 1:3000 dilution with Enzyme conjugate diluent) per well was added to the plate and incubated at room temperature for 30 minutes. The plate was washed, and 50 uL of TMB substrate per well was added and incubated at room temperature for 5 minutes. 50 uL of stop solution per well was added. The wells were read at 450 nm. The results are shown in FIG. 16.

[0194] Figure 16A presents the results of the assessment of the extended peptide of D678 in an indirect ELISA format using a time course, rabbit pAb, and SPF cats. The synthetic peptides D678 amino and D678 carboxyl showed similar reactivity to samples from the time course samples, suggesting that the extended region did not increase the immunogenicity of the peptide. The rabbit pAb against TDX1557 did not show any antibody response to the peptide D678, whereas the rabbit pAb reacted with both the extended peptides D678 amino and D678 carboxyl. The peptides D678, D678 amino, and D678 carboxyl did not show any significant reactivity to serum samples from experimentally infected cats.

[0195] Example 11 Detection of lungworm antigen (2M2 antigen) in fecal samples using rabbit polyclonal antibody against TDX1557 In this example, fecal samples collected from an experimentally infected cat (Big Mac) at 36 days, 64 days, 141 days, and 180 days after infection were mixed with SNAP® Giardia Dilution (IDEXX Laboratories, Westbrook, ME) at a ratio of 10% (0.5 g sample in 5 ml diluent). The suspension was mixed and centrifuged at 10,000 g for 5 minutes. 100 ul of 2×SDS-PAGE sample buffer was added to 100 ul of the supernatant, and the mixture was kept in a heating block (90° C.) for 10 minutes. The mixture was subjected to SDS-PAGE electrophoresis using routine procedures. The resulting gel was subjected to immunoblot analysis by transferring the total proteins of SDS-PAGE to a nitrocellulose (NC) membrane. The NC membrane was probed with rabbit polyclonal antibody against TDX1557 followed by addition of alkaline phosphatase conjugate (alkaline phosphatase coupled anti-rabbit IgG H+L, Sigma, St. Louis, MO). Blots were developed with BCIP / NBT substrate (Sigma, St. Louis, MO). The positive control is rTDX1557. The negative control is a fecal sample from a healthy uninfected cat. FIG. 17 shows the detection of A. abstrusus antigen (2M2) in fecal samples from BigMac, an experimentally infected cat. The results confirm the detection of a 62 kDA band in fecal samples from BigMac at 36 days post-infection, 64 days post-infection, and 141 days post-infection using rabbit polyclonal antibody against TDX1557. No band was detectable at 180 days post-infection. Experimentally infected cats were treated with antiparasitic drugs 90 days after infection, and 2M2 antigen was detectable in fecal samples up to 140 days after infection. However, 2M2 antigen disappeared completely by 180 days after infection. The positive control was recombinant 2M2 antigen (rTDX1557), and the negative control was a fecal sample from a healthy, uninfected cat.

[0196] Example 12 Alanine scanning of the epitope 2M2 This example shows the effect of amino acid substitutions on antibody binding within the region defined by peptides D6, D7, and D8. For this purpose, an alanine scan was performed. Alanine (bold) was substituted for the native amino acids in peptide D7M (SEQ ID NO: 90) and in peptide D8M (SEQ ID NO: 106) as shown in the table below. In positions where alanine was already present in the native sequence, it was substituted with glycine.

[0197] [Table 3]

[0198] [Table 4]

[0199] The peptide was synthesized and labelled with N-terminal biotin using standard procedures.

[0200] If a significant drop in OD value is observed, amino acids critical for antibody binding can be detected, indicating that the substituted amino acid was necessary for proper antibody binding. In addition, this experiment provides an opportunity for the identification of favorable substitutions where the OD value is increased compared to the original peptide.

[0201] Indirect ELISA assays examining the immunoreactivity of peptides D7M0-D7M15 and D8M0-D8M10 with pooled sera from three cats naturally infected with A. abstrusus (i.e., field positive cats) were performed as described in Example 7A. Assays were performed in triplicate. OD values ​​were normalized to the immunoreactivity (OD value) with sera from day 0 samples of experimentally infected cats.

[0202] The results are shown in Figure 18. The strong reduction in OD values ​​by D8M7 (tryptophan to alanine) and D8M8 (phenylalanine to alanine) compared to D8M0 indicates that the amino acids at positions 7 and 8 are crucial for binding of antibodies in serum from cats infected with A.abstrusus. The increase in OD values ​​by D8M10 compared to D8M0 indicates that the substitution of glutamic acid (a negatively charged amino acid) with alanine (a small non-polar amino acid) enhanced the binding of the peptide to antibodies in serum from cats infected with A.abstrusus. Similar results were obtained when the peptide was examined with serum from cats experimentally infected with A.abstrusus at various time points (data not shown).

[0203] Thus, the amino acids corresponding to positions 7 (tryptophan) and 8 (phenylalanine) of peptide D8M0 (SEQ ID NO: 106) are critical for binding to antibodies from cats infected with A. abstrusus. These amino acid positions correspond to positions 4 (tryptophan) and 5 (phenylalanine) of the peptide having the sequence SKSWF (SEQ ID NO: 81).

[0204] This data confirms that the consensus sequence for the core epitope is XXXWF (SEQ ID NO: 117). Thus, in one embodiment of the disclosure, the polypeptide comprises XXXWF (SEQ ID NO: 117), in which: (a) the amino acids W and F at positions 4 and 5, respectively, are retained, and at least one X at positions 1, 2, and 3 is substituted with a conservative or non-conservative amino acid; (b) the amino acids W and F at positions 4 and 5, respectively, are retained, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) the amino acids W and F at positions 4 and 5, respectively, are retained, and X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid; (d) the amino acid W at position 4 or the amino acid F at position 5 is substituted with a conservative amino acid, in which X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid.

[0205] Some embodiments of the present disclosure include the following.

[0206] Embodiment 1: Amino acid sequences of SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d2 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d4 peptide), SEQ ID NO:38 (d5 peptide), SEQ ID NO:39 (d678 peptide), SEQ ID NO:40 (d15 peptide), SEQ ID combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino-extended), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), A polypeptide comprising SEQ ID NO:115 (D8M9), SEQ ID NO:116 (D8M10), or XXXWF (SEQ ID NO:117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid;

[0207] Embodiment 2: Amino acid sequences of SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d20 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d20 peptide), SEQ ID NO:35 (d35 peptide), SEQ ID NO:36 (d36 peptide), SEQ ID NO:37 (d4 peptide), SEQ ID NO:38 (d5 peptide), SEQ ID NO:39 (d678 peptide), SEQ ID NO:40 (d15 peptide), SEQ combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D 8M6), SEQ ID NO:115 (D8M9), SEQ ID NO:116 (D8M10), or XXXWF (SEQ ID NO:117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4 or the amino acid F at position 5 is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid;

[0208] Embodiment 3: Amino acid sequences of SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage), SEQ ID NO:5 (T3 cleavage), SEQ ID NO:6 (T4 cleavage), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d15 peptide), SEQ ID NO:33 (d16 peptide), SEQ ID NO:34 (d17 peptide), SEQ ID NO:35 (d18 peptide), SEQ ID NO:36 (d19 peptide), SEQ ID NO:37 (d15 peptide), SEQ ID NO:38 (d15 peptide), SEQ ID NO:39 (d16 peptide), SEQ ID NO:40 (d17 peptide), S combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:1 15 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative amino acid or a non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid;

[0209] Embodiment 4: The polypeptide of embodiment 1, which is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 amino acids, or full length, of TDX1557. Alternatively, the polypeptide of embodiment 1, which comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids set forth in SEQ ID NOs: 2-18 and 78-117.

[0210] Embodiment 5: A peptide having 10 to 25 linked amino acids, comprising SEQ ID NO:2 (TDX1557), SEQ ID NO:3 (rTDX1557), SEQ ID NO:4 (T2 cleavage product), SEQ ID NO:5 (T3 cleavage product), SEQ ID NO:6 (T4 cleavage product), SEQ ID NO:7 (C10 peptide), SEQ ID NO:8 (C11 peptide), SEQ ID NO:9 (C12 peptide), SEQ ID NO:10 (D1 peptide), SEQ ID NO:11 (D2 peptide), SEQ ID NO:12 (D3 peptide), SEQ ID NO:13 (D4 peptide), SEQ ID NO:14 (D5 peptide), SEQ ID NO:15 (D6 peptide), SEQ ID NO:16 (D7 peptide), SEQ ID NO:17 (D8 peptide), SEQ ID NO:18 (d678 peptide), SEQ ID NO:19 (d78 peptide), SEQ ID NO:20 (d8 peptide), SEQ ID NO:21 (d9 peptide), SEQ ID NO:22 (d10 peptide), SEQ ID NO:23 (d11 peptide), SEQ ID NO:24 (d12 peptide), SEQ ID NO:25 (d13 peptide), SEQ ID NO:26 (d14 peptide), SEQ ID NO:27 (d15 peptide), SEQ ID NO:28 (d16 peptide), SEQ ID NO:29 (d17 peptide), SEQ ID NO:30 (d18 peptide), SEQ ID NO:31 (d19 peptide), SEQ ID NO:32 (d2 peptide), SEQ ID NO:33 (d20 peptide), SEQ ID NO:34 (d15 peptide), SEQ ID NO:35 (d16 peptide), SEQ ID NO:36 (d17 peptide), SEQ ID NO:37 (d18 peptide), SEQ ID NO:38 (d19 peptide), SEQ ID NO:39 (d20 peptide), SEQ combo), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678: amino-extended), SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 (D8M0), SEQ ID NO:107 (D8M1 peptide), SEQ ID NO:108 (D8M2), SEQ ID NO:109 (D8M3), SEQ ID NO:110 (D8M4), SEQ ID NO:111 (D8M5), SEQ ID NO:112 (D8M6), SEQ ID NO:115 (D8M9 ), at least 5, 6, 7, 8, 9, or 10 consecutive amino acids as set forth in SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in which: (a) the amino acids W and F in positions 4 and 5, respectively, are retained and at least one X in positions 1, 2, and 3 is substituted with a conservative or non-conservative amino acid; (b) the amino acids W and F in positions 4 and 5, respectively, are retained and conservative amino acid substitutions of X are made in positions 1, 2, and 3; (c) the amino acids W and F in positions 4 and 5, respectively, are retained and X in positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X in position 2 is the amino acid K, a conservative amino acid, or a non-conservative amino acid;(d) the amino acid W at position 4, or the amino acid F at position 5, is substituted with a conservative amino acid, where X at positions 1 and 3 are independently the amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid;

[0211] Embodiment 6: The polypeptide of embodiment 1, wherein the peptide is labeled or conjugated to a solid support.

[0212] Embodiment 7: A method for detecting the presence or absence of one or more Leptinus felidae antibodies in a biological sample from a mammal, comprising: (a) contacting the sample with one or more polypeptides according to any one of embodiments 1 to 6, which are capable of specifically binding to one or more Leptinus cati antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample, if present; and (c) detecting the presence or absence of an antibody-polypeptide complex, if any. The method includes:

[0213] Embodiment 8: A method for diagnosing whether a mammal is infected with one or more lungworms, comprising: (a) contacting a biological sample derived from a mammal with one or more polypeptides according to any one of embodiments 1 to 6, which are capable of specifically binding to one or more Leptinus cati antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample, if present; (c) detecting the presence or absence of an antibody-polypeptide complex, if any; and (d) diagnosing the mammal as having feline lungworm if the antibody-polypeptide complex is present. The method includes:

[0214] Embodiment 9: A method of diagnosing and treating a mammal infected with one or more lungworms, comprising: (a) contacting a biological sample derived from a cat with one or more polypeptides according to any one of embodiments 1 to 6, which are capable of specifically binding to one or more antibodies against Leptinus felidae; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample, if present; (c) detecting the presence or absence of an antibody-polypeptide complex, if any; (d) diagnosing the mammal as having L. felineus if the antibody-polypeptide complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat a mammal having a feline lungworm infection. The method includes:

[0215] Embodiment 10: A method for detecting the presence or absence of one or more feline lungworm antigens in a biological sample, comprising: (a) contacting a sample from a cat with one or more antibodies that specifically bind to one or more polypeptides of any one of embodiments 1 to 6; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if present, in the sample; and (c) detecting the presence or absence of an antibody-antigen complex, if any. The method includes:

[0216] Embodiment 11: A method for diagnosing whether a mammal is infected with one or more lungworms, comprising: (a) contacting a biological sample derived from a mammal with one or more antibodies that specifically bind to one or more polypeptides of any one of embodiments 1 to 6; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if present, in the sample; (c) detecting the presence or absence of an antibody-antigen complex, if any; and (d) diagnosing the mammal as having feline lungworm if the antibody-antigen complex is present. The method includes:

[0217] Embodiment 12: A method of diagnosing and treating a mammal infected with one or more lungworms, comprising: (a) contacting a biological sample derived from a mammal with one or more antibodies that specifically bind to one or more polypeptides of any one of embodiments 1 to 6; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if present, in the sample; (c) detecting the presence or absence of an antibody-antigen complex, if any; (d) diagnosing the mammal as having L. felineus if the antibody-antigen complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat a mammal having a feline lungworm infection. The method includes:

[0218] Embodiment 13: The method of embodiment 9 or 12, wherein step (e) further comprises one or more additional therapeutic agents to treat infection by one or more helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria.

[0219] Embodiment 14: The method of any one of embodiments 8, 9, 11, or 12, wherein (b) step (d) further comprises one or more therapeutic agents to control, eradicate, or kill an intermediate host of a feline lungworm parasite, a flatworm parasite, a helminth parasite, a non-helminth parasite, a virus, a fungus, a protozoa, or a bacterium.

[0220] Embodiment 15: The method of any one of embodiments 1 to 14, wherein the one or more antibodies bind to Aelurostrongylus abstrusus but do not bind to antibodies against Capillaria aerophila or Troglostrongylus brevior.

[0221] Embodiment 16: The method of embodiments 7 to 9, wherein the one or more polypeptides do not bind to an antibody against a roundworm, tapeworm, whipworm, or hookworm.

[0222] Embodiment 17: The method of embodiments 10 to 12, wherein the one or more antibodies do not bind to an antigen of a roundworm, a tapeworm, a whipworm, or a hookworm.

[0223] Embodiment 18: The method of embodiment 16 or 17, wherein the roundworm is of the genus Toxocara.

[0224] Embodiment 19: The method of any one of embodiments 16 to 18, wherein the tapeworm is of the genus Taenia or Diphylidium.

[0225] Embodiment 20: The method of any one of embodiments 16 to 19, wherein the hookworm is of the genus Ancylostoma.

[0226] Embodiment 21: The method of any one of embodiments 7 to 20, wherein the step (c) of detecting the presence or absence of the complexes further comprises the step of providing at least one secondary antibody that binds to the one or more complexes.

[0227] Embodiment 22: The method of embodiment 21, wherein the secondary antibody is labeled or conjugated to a solid support.

[0228] Embodiment 23: The method of embodiment 22, wherein the solid support forms part of an enzyme-linked immunosorbent assay device.

[0229] Embodiment 24: The method of embodiment 23, wherein the enzyme-linked immunosorbent assay device is a lateral flow immunoassay device.

[0230] Embodiment 25: The method of any one of embodiments 1 to 24, wherein the mammal is a cat.

[0231] Embodiment 26: A polynucleotide encoding a polypeptide according to any one of embodiments 1 to 6.

[0232] Embodiment 27: The polynucleotide of embodiment 26, which is RNA.

[0233] Embodiment 28: The polynucleotide of embodiment 27, comprising one or more modified uridine residues.

[0234] Embodiment 29: The polynucleotide of embodiment 28, wherein the modified uridine residue is pseudouridine or methylpseudouridine.

[0235] Embodiment 30: The polynucleotide of embodiment 26, which is an mRNA.

[0236] Embodiment 31: The polynucleotide of embodiment 30, contained within a lipid nanoparticle.

[0237] Embodiment 32: A vaccine composition comprising one or more polynucleotides according to any one of embodiments 26 to 31 and a pharma- ceutically acceptable carrier.

[0238] Embodiment 33: A vaccine composition comprising one or more polypeptides according to any one of embodiments 1 to 6 and a pharma- ceutically acceptable carrier.

[0239] Embodiment 34: An immune complex comprising one or more polypeptides according to any one of embodiments 1 to 6 and one or more antibodies against L. felineus, specifically bound to the one or more polypeptides.

[0240] Embodiment 35: An immune complex comprising one or more antibodies that specifically bind to one or more polypeptides according to any one of embodiments 1 to 6, and one or more feline lungworm antigens specifically bound to the one or more antibodies.

[0241] Embodiment 36: A device for detecting the presence or absence of one or more antibodies against feline lungworm from a biological sample, comprising a solid support, on which one or more polypeptides according to any one of embodiments 1 to 6 are immobilized.

[0242] Embodiment 37: The device of embodiment 36, wherein the one or more polypeptides do not specifically cross-react with one or more antibodies against roundworms, tapeworms, hookworms, Capillaria aerophila, or Troglostrongylus brevior.

[0243] Embodiment 38: A device according to embodiment 37, wherein the roundworm is of the genus Toxocara, and / or the tapeworm is of the genus Taenia or Diphylidium, and / or the hookworm is of the genus Ancylostoma.

[0244] Embodiment 39: A device for detecting the presence or absence of one or more feline lungworm antigens from a biological sample, comprising a solid support on which one or more antibodies are immobilized that specifically bind to one or more polypeptides according to any one of embodiments 1 to 6.

[0245] Embodiment 40: The device of embodiment 39, wherein the one or more antibodies do not specifically cross-react with one or more antigens of roundworms, tapeworms, hookworms, Capillaria aerophila, or Troglostrongylus brevior.

[0246] Embodiment 41: A device according to embodiment 40, wherein the roundworm is of the genus Toxocara, and / or the tapeworm is of the genus Taenia or Diphylidium, and / or the hookworm is of the genus Ancylostoma.

[0247] Embodiment 42: A method of immunizing a mammal, such as a cat, against Lungworm, comprising administering a vaccine composition according to embodiment 32 or 33.

Claims

1. A method for detecting the presence or absence of one or more feline lungworm antibodies in a biological sample derived from a mammal, (a) A step of contacting a sample with one or more polypeptides capable of specifically binding to one or more feline lungworm antibodies, wherein the polypeptides are amino acid sequences such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) the step of forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample, if present; and (c) A step to detect the presence or absence of the antibody-polypeptide complex, if present. A method that includes this.

2. A method for diagnosing whether a mammal is infected with one or more feline lungworms, (a) A step of contacting a biological sample derived from a mammal with one or more polypeptides capable of specifically binding to one or more feline lungworm antibodies, wherein the polypeptide is an amino acid sequence such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) If present, the step of forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample; (c) A step of detecting the presence or absence of an antibody-polypeptide complex, if present; and (d) A step of diagnosing a mammal having feline lungworm in the presence of an antibody-polypeptide complex. A method that includes this.

3. A method for diagnosing and treating a mammal infected with one or more feline lungworms, (a) A step of contacting a biological sample derived from a mammal with one or more polypeptides capable of specifically binding to one or more feline lungworm antibodies, wherein the polypeptide is an amino acid sequence such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) If present, the step of forming an antibody-polypeptide complex in the presence of one or more antibodies in the sample; (c) A step of detecting the presence or absence of an antibody-polypeptide complex, if present; (d) The step of diagnosing a mammal having feline lungworm in the presence of an antibody-polypeptide complex; and (e) A step of treating a mammal infected with feline pulmonary nematode by administering an effective dose of one or more therapeutic agents. A method that includes this.

4. A method for detecting the presence or absence of one or more feline lungworm antigens in a biological sample, (a) A step of contacting a mammalian sample with one or more antibodies that specifically bind to one or more polypeptides, wherein the polypeptide is an amino acid sequence such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) If present, the step of forming an antibody-antigen complex in the presence of one or more feline lungworm antigens in the sample; and (c) A step to detect the presence or absence of an antibody-antigen complex, if present. A method that includes this.

5. A method for diagnosing whether a mammal is infected with one or more feline lungworms, (a) A step of contacting a biological sample derived from a mammal with one or more antibodies that specifically bind to one or more polypeptides, wherein the polypeptide is an amino acid sequence such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) If present, the step of forming an antibody-antigen complex in the presence of one or more feline lungworm antigens in the sample; (c) A step of detecting the presence or absence of an antibody-antigen complex, if present; and (d) A step to diagnose a mammal having feline lungworm if an antibody-antigen complex is present. A method that includes this.

6. A method for diagnosing and treating a mammal infected with one or more feline lungworms, (a) A step of contacting a biological sample derived from a mammal with one or more antibodies that specifically bind to one or more polypeptides, wherein the polypeptide is an amino acid sequence such as SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M 6) Sequence ID 115 (D8M9), Sequence ID 116 (D8M10), or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The step comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid; (b) If present, the step of forming an antibody-antigen complex in the presence of one or more feline lungworm antigens in the sample; (c) A step of detecting the presence or absence of an antibody-antigen complex, if present; (d) The step of diagnosing a mammal having feline lungworm in the presence of an antibody-antigen complex; and (e) A step of treating a mammal infected with feline pulmonary nematode by administering an effective dose of one or more therapeutic agents. A method that includes this.

7. The method according to claim 3 or 6, wherein step (e) further comprises one or more further therapeutic agents for treating an infection caused by (a) one or more parasitic helminths, one or more non-helminth parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria.

8. The method according to any one of claims 2, 3, 5, or 6, wherein step (d) further comprises one or more therapeutic agents, and (b) control, eliminate, or kill an intermediate host of a parasite which is a feline lung nematode, a flatworm helminth parasite, a parasitic helminth parasite, a parasitic helminth other than a helminth parasite, a virus, a fungus, a protozoan, or a bacterium.

9. The method according to any one of claims 1 to 6, wherein one or more antibodies bind to Aelurostrongylus abstrusus but do not bind to antibodies against Capillaria aerophila or Troglobistrongylus brevior.

10. The method according to claims 1 to 3, wherein one or more polypeptides do not bind to antibodies against roundworms, tapeworms, whipworms, or hookworms.

11. The method according to claims 4 to 6, wherein one or more antibodies do not bind to antigens of roundworms, tapeworms, whipworms, or hookworms.

12. The method according to claim 10, wherein the roundworm is of the genus Toxocara, and / or the tapeworm is of the genus Taenia or Diphyllidium, and / or the hookworm is of the genus Ancylostoma.

13. The method according to any one of claims 1 to 6, wherein the step (c) of detecting the presence or absence of a complex further comprises the step of preparing at least one secondary antibody that binds to one or more complexes.

14. The method according to claim 13, wherein the secondary antibody is labeled or bonded to a solid support.

15. The method according to claim 14, wherein the solid support forms part of an enzyme-coupled immunosorbent assay device.

16. The method according to claim 15, wherein the enzyme-linked immunosorbent assay device is a lateral flow immunoassay device.

17. The method according to any one of claims 1 to 6, wherein the mammal is a cat.

18. The polypeptide is an amino acid sequence: SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D 8M6), SEQ ID NO: 115 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2, and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2, and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The method according to any one of claims 1 to 6, selected from the group comprising polypeptides including: (d) amino acid W at position 4 or amino acid F at position 5 is substituted with a conserved amino acid, in which case X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid.

19. The polypeptide is an amino acid sequence: SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0 peptide), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), Sequence No. Of sequence 115 (D8M9) and sequence number 116 (D8M10), at least five consecutive amino acids, or XXXWF (sequence number 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;The method according to any one of claims 1 to 6, comprising a polypeptide containing (d) amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid.

20. The method according to any one of claims 1 to 6, wherein the polypeptide is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 amino acids or full-length amino acids from TDX1557.

21. The method according to any one of claims 1 to 6, wherein the polypeptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids as specified in SEQ ID NOs. 2 to 18 and 78 to 117.

22. The polypeptide has 10 to 25 linked amino acids, and includes SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0 peptide), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), SEQ ID NO: 115 (D8 M9), as specified in Sequence ID 116 (D8M10), at least 5, 6, 7, 8, 9, or 10 consecutive amino acids, or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2, and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2, and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;The method according to claim 20, comprising a polypeptide containing (d) amino acid W at position 4 or amino acid F at position 5, wherein X at positions 1 and 3 is independently amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid.

23. The method according to any one of claims 1 to 6, wherein the peptide is labeled or bonded to a solid support.

24. The amino acid sequences are: SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), Polypeptides containing SEQ ID NO: 115 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2, and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2, and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) A polypeptide comprising: amino acid W at position 4, or amino acid F at position 5, which is substituted with a conserved amino acid, in which case X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid;

25. The amino acid sequences are: SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D 8M6), SEQ ID NO: 115 (D8M9), SEQ ID NO: 116 (D8M10), or XXXWF (SEQ ID NO: 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2, and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2, and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The polypeptide according to claim 24, selected from the group comprising polypeptides including amino acid W at position 4 or amino acid F at position 5, in which case X at positions 1 and 3 are independently amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid.

26. The amino acid sequences are: SEQ ID NO: 2 (TDX1557), SEQ ID NO: 3 (rTDX1557), SEQ ID NO: 4 (T2 cleavage), SEQ ID NO: 5 (T3 cleavage), SEQ ID NO: 6 (T4 cleavage), SEQ ID NO: 7 (C10 peptide), SEQ ID NO: 8 (C11 peptide), SEQ ID NO: 9 (C12 peptide), SEQ ID NO: 10 (D1 peptide), SEQ ID NO: 11 (D2 peptide), SEQ ID NO: 12 (D3 peptide), SEQ ID NO: 13 (D4 peptide), SEQ ID NO: 14 (D5 peptide), SEQ ID NO: 15 (D6 peptide), SEQ ID NO: 16 (D7 peptide), SEQ ID NO: 17 (D8 peptide), SEQ ID NO: 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0 peptide), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), Sequence No. Of sequence 115 (D8M9) and sequence number 116 (D8M10), at least five consecutive amino acids, or XXXWF (sequence number 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2 and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2 and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The polypeptide according to claim 24, comprising a polypeptide having amino acid W at position 4 or amino acid F at position 5, in which case X at positions 1 and 3 are independently amino acid S, a conservative amino acid, or a non-conservative amino acid, and X at position 2 is K, a conservative amino acid, or a non-conservative amino acid.

27. The polypeptide according to claim 24, wherein at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids, or full-length amino acids, are selected from TDX1557.

28. The polypeptide according to claim 24, comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acids as specified in SEQ ID NOs. 2-18 and 78-117.

29. It has 10 to 25 linked amino acids, and is the sequence number 2 (TDX1557), sequence number 3 (rTDX1557), sequence number 4 (T2 cleavage), sequence number 5 (T3 cleavage), sequence number 6 (T4 cleavage), sequence number 7 (C10 peptide), sequence number 8 (C11 peptide), sequence number 9 (C12 peptide), sequence number 10 (D1 peptide), sequence number 11 (D2 peptide), sequence number 12 (D3 peptide), sequence number 13 (D4 peptide), sequence number 14 (D5 peptide), sequence number 15 (D6 peptide), sequence number 16 (D7 peptide), sequence number 17 (D8 peptide), sequence number 18 (d678 combo), SEQ ID NO: 78 (modified d678), SEQ ID NO: 79 (d678: amino extended form), SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 (D8M0 peptide), SEQ ID NO: 107 (D8M1 peptide), SEQ ID NO: 108 (D8M2), SEQ ID NO: 109 (D8M3), SEQ ID NO: 110 (D8M4), SEQ ID NO: 111 (D8M5), SEQ ID NO: 112 (D8M6), SEQ ID NO: 115 (D8 M9), as specified in Sequence ID 116 (D8M10), at least 5, 6, 7, 8, 9, or 10 consecutive amino acids, or XXXWF (Sequence ID 117) [in the sequence, (a) amino acids W and F at positions 4 and 5 are retained, respectively, and at least one X at positions 1, 2, and 3 is substituted with a conserved or non-conserved amino acid; (b) amino acids W and F at positions 4 and 5 are retained, respectively, and the conserved amino acid substitution of X is made at positions 1, 2, and 3; (c) amino acids W and F at positions 4 and 5 are retained, respectively, and X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is amino acid K, a conserved amino acid, or a non-conserved amino acid;(d) The polypeptide according to claim 26, comprising a polypeptide in which amino acid W at position 4 or amino acid F at position 5 is substituted with a conserved amino acid, in which case X at positions 1 and 3 is independently amino acid S, a conserved amino acid, or a non-conserved amino acid, and X at position 2 is K, a conserved amino acid, or a non-conserved amino acid.

30. The polypeptide according to claim 24, wherein the peptide is labeled or bonded to a solid support.

31. A polynucleotide encoding a polypeptide according to any one of claims 24 to 29.

32. The polynucleotide according to claim 31, which is RNA.

33. The polynucleotide according to claim 32, comprising one or more modified uridine residues.

34. The polynucleotide according to claim 33, wherein the modified uridine residue is pseudouridine or methylpseudridine.

35. The polynucleotide according to claim 31, which is mRNA.

36. The polynucleotide according to claim 35, contained within lipid nanoparticles.

37. A vaccine composition comprising one or more polynucleotides according to claim 31 and a pharmaceutically acceptable carrier.

38. A vaccine composition comprising one or more polypeptides according to any one of claims 24 to 30 and a pharmaceutically acceptable carrier.

39. An immune complex comprising one or more polypeptides according to any one of claims 24 to 30 and one or more feline lungworm antibodies specifically bound to one or more polypeptides.

40. An immune complex comprising one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 24 to 30, and one or more feline pulmonary nematode antigens specifically bound to one or more antibodies.

41. A device for detecting the presence or absence of one or more feline lungworm antibodies derived from a biological sample, comprising a solid support on which one or more polypeptides according to any one of claims 24 to 30 are immobilized.

42. The device according to claim 41, wherein one or more polypeptides do not specifically cross-react with one or more antibodies against roundworms, tapeworms, hookworms, Capillaria aerophila, or Troglobistrungylus brevior.

43. The device according to claim 42, wherein the roundworm is of the genus Toxocara, and / or the tapeworm is of the genus Taenia or Diphyllidium, and / or the hookworm is of the genus Ancylostoma.

44. A device for detecting the presence or absence of one or more feline lungworm antigens derived from a biological sample, comprising a solid support on which one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 24 to 30 are immobilized.

45. The device according to claim 44, wherein one or more antibodies do not specifically cross-react with one or more antigens of roundworms, tapeworms, hookworms, Capillaria aerophila, or Troglobistrungylus brevior.

46. The device according to claim 45, wherein the roundworm is of the genus Toxocara, and / or the tapeworm is of the genus Taenia or Diphyllidium, and / or the hookworm is of the genus Ancylostoma.

47. A kit comprising the device described in claim 41.

48. A method for immunizing a mammal against feline lungworm, comprising the step of administering the vaccine composition described in claim 37.

49. A kit comprising the device described in claim 44.

50. A method for immunizing a mammal against feline lungworm, comprising the step of administering the vaccine composition described in claim 38.

51. The method according to claim 11, wherein the roundworm is of the genus Toxocara and / or the tapeworm is of the genus Taenia or Diphyllidium and / or the hookworm is of the genus Ancylostoma.

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