Methods, devices, kits and compositions for detecting feline lung nematodes

Abstract

Compositions, devices, kits, and methods are provided for the detection of A.abstruus of the feline lung nematode species, i.e., A.abstruus, in a mammal. More specifically, polypeptides and polypeptide compositions, antibodies and antibody compositions, devices, kits and methods are provided, the polypeptides and polypeptide compositions, antibodies and antibody compositions, devices, kits, and methods are useful for detecting the presence or absence of strongylus felis antibodies in a sample from a mammal that may also comprise one or more other feline lung nematode, roundworm, hookworm, and whipworm antigens; the kit is used for diagnosing felstrongylus felina lung nematode infection; and for the detection, diagnosis and treatment of catrostrongylus felina lung nematode infection. Compositions, vaccines, and methods comprising the polypeptide or polynucleotide immunogen for treating and / or preventing feline lung nematode infection in a mammal are also provided.

Description

Cross-references

[0001] This application claims priority to U.S. Provisional Application No. 63 / 323,558, filed on March 25, 2022, which is incorporated herein by reference in its entirety. Statement regarding sequence listing

[0002] The sequence listing in computer readable form is submitted with this application by electronic submission and is incorporated by reference in its entirety. The sequence listing submitted with this application is contained in a text file named "20-1518-WO_Sequence-Listing.xml" created on March 21, 2023 and is 166,470 bytes in size. Technical Field

[0003] The present disclosure relates to compositions, devices, kits and methods for detecting the cat lung nematode species Aelurostronglylus abstrus ("A. abstrusus") in mammals; and compositions for treating and / or preventing cat lung nematode infections in mammals and vaccines comprising the compositions. More specifically, the present disclosure relates to polypeptides and polypeptide compositions, antibodies and antibody compositions, devices, kits and methods for detecting the presence or absence of A. abstrus antibodies in samples from mammals that may also contain one or more other cat lung nematode, roundworm, hookworm and whipworm antigens; and polypeptides and polynucleotides for treating and / or preventing cat lung nematode infections in mammals and vaccines comprising the polypeptides or polynucleotides. Background Art

[0004] Feline lung nematode disease (aelurostrongylosis) is a parasitic lung disease of veterinary importance. Strongylus cati is the most famous cat lung nematode and is considered to be the most common in domestic cats worldwide. Other cat lung nematodes include Oslerus rostratus ("O.rostratus"), Troglostrongylus brevior ("T.brevior"), Capillaria aerophila, and Paragonimus spp. The nematode Strongylus cati has an indirect life cycle, which includes cats as definitive hosts, terrestrial molluscs as intermediate hosts, and small vertebrates (e.g., rodents, birds, lizards, and frogs) as relay hosts. Adult worms are located in the alveolar ducts and bronchioles of cat hosts. Female worms hatch eggs in the lung parenchyma and small blood vessels, where the first stage (L1) larvae develop. L1 larvae migrate to the throat via the bronchi and trachea, where they are coughed up, swallowed, and enter the environment through feces. L1 larvae develop into infective L3 larvae, enter their immediate host, and are then picked up by the secondary host. Ingestion of the secondary host by the feline host is the most recognized mode of lungworm transmission. Once the adult worms inhabit the lungs of the feline host, they cause respiratory symptoms ranging from mild respiratory symptoms to interstitial bronchopneumonia, dyspnea, and respiratory distress, which may lead to death, especially in young, debilitated, or immunosuppressed cats. This nonspecific clinical pattern often requires a high level of clinical awareness of the disease to initiate prompt treatment.

[0005] In addition to the lack of specific clinical signs, a major concern regarding the treatment and prevention of feline lungstrongylosis is the lack of a clear diagnostic procedure. Current methods for diagnosing Strongyloidiasis in cats primarily involve microscopic examination of fecal samples, either directly in a fecal smear or after concentrating the eggs and parasites by flotation in a density medium or by sedimentation. Despite the widespread adoption of this procedure, the method has significant drawbacks. These microscopic methods are time-consuming and require specialized equipment. Furthermore, the accuracy of the results from these methods is highly dependent on the skill and expertise of the operator. For example, the Baermann method is considered the standard method for Strongyloidiasis diagnosis and involves the recovery of larvae from feces. However, the Baermann method may show false-negative results due to the low concentration of larvae in fecal samples. Furthermore, this technique requires specific skills to distinguish Strongyloidiasis larvae from other lungworm larvae, as Strongyloidiasis, O. rostrotus, and Strongyloides brevis may result in mixed infections, as they share the same intermediate and secondary hosts. Not surprisingly, Strongyloidiasis in cats is often misdiagnosed or missed during routine fecal examinations.

[0006] Feces handling is unpleasant and dangerous. The hygienic and harmless procedures for handling feces are inconvenient and often complicated. These procedures may include weighing, centrifugation and storage, which are difficult to complete unless performed in a clinical laboratory equipped with appropriate equipment, protective equipment and skilled technicians. Therefore, any reduction in the number of steps required for fecal testing and any reduction in contact between the test operator and the test material are desirable. Clinical laboratories have been using immunoassays to detect various viruses, bacteria, and non-helminth parasites and organisms in feces. However, there is still a need for a simple immunoassay for detecting cat nematode infections in feces, whole blood or serum. In addition, there is still a need to treat and / or prevent cat lung nematode (e.g., cat nematode) infections in mammals. Summary of the invention

[0007] In one aspect, the disclosure includes polypeptides comprising an epitope of a Strongyloides felis antigen. In one embodiment, the polypeptide comprises the following amino acid sequence: 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 combination), SEQ ID NO:78 (modified D678), SEQ ID NO:79 (D678-amino extension), 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, SEQID 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 XXXWF (SEQ ID NO:117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and making conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids, as listed herein; or a polypeptide comprising a sequence that 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 a Strongyloides felis antigen. In one embodiment, the combination polypeptide comprises one or more polypeptides comprising the following amino acid sequence: 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (D678-amino extension), SEQ ID NO: 80 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 (D8MO 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 (D8M9), SEQ ID NO: 116 (D8M10);Or a polypeptide comprising XXXWF (SEQ ID NO: 117), wherein (a) 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 replaced with a conservative or non-conservative amino acid; (b) 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) amino acids W and F at positions 4 and 5, respectively, are retained, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids. In another embodiment, the combination polypeptide comprises two or more polypeptides having the following amino acid 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, SEQ ID NO:17, SEQ ID NO:78 or SEQ ID NO:79; or variants thereof that differ only in conservative substitutions and / or modifications. The combination polypeptide comprises at least one epitope of a cat nematode antigen having the amino acid sequences 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 can be prepared by synthetic methods or using recombinant DNA technology. ;

[0009] In another aspect, the disclosure includes antibodies that specifically bind to a polypeptide comprising all or an antigenic portion of an amino acid sequence corresponding to one or more of the following 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D778), SEQ ID NO: 80 (modified D778), SEQ ID NO: 81 (modified D778), SEQ ID NO: 82 (modified D778), SEQ ID NO: 83 (modified D778), SEQ ID NO: 84 (modified D778), SEQ ID NO: 85 (modified D778), SEQ ID NO: 86 (modified D778), SEQ ID NO: 87 (modified D778), SEQ ID NO: 88 (modified D778), SEQ ID NO: 89 (modified D778), SEQ ID NO: 90 (modified D778), SEQ ID NO: 91 (modified D778), SEQ ID NO: 92 (modified D778 NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2 and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2 and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acid or non-conservative amino acid; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acid or non-conservative amino acid, as listed herein; or a polypeptide comprising a sequence that is a conservative variant of one of these sequences. In a further aspect, the antibody specifically binds to an antigen from a mammal infected with Strongyloides felis, but does not specifically bind to an antigen from a mammal infected with hookworms, roundworms, whipworms, heartworms, and other feline lung nematodes.;

[0010] In another aspect, the disclosure includes antibodies obtained by immunization with a polypeptide comprising all or an antigenic portion of an amino acid sequence corresponding to one or more of the following 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D778), SEQ ID NO: 80 (modified D778), SEQ ID NO: 81 (modified D778), SEQ ID NO: 82 (modified D778), SEQ ID NO: 83 (modified D778), SEQ ID NO: 84 (modified D778), SEQ ID NO: 85 (modified D778), SEQ ID NO: 86 (modified D778), SEQ ID NO: 87 (modified D778), SEQ ID NO: 88 (modified D778), SEQ ID NO: 89 (modified D778), SEQ ID NO: 90 (modified D778), SEQ ID NO: 91 (modified D778), SEQ ID NO: 92 (modified D7 NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2 and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2 and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; or a polypeptide comprising a sequence that is a conservative variant of one of these sequences. ;

[0011] In these related aspects, DNA sequences encoding the above-mentioned polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided.

[0012] In yet another aspect, the present disclosure provides a device for detecting the presence or absence of a cat roundworm antigen in a sample; the device comprises a solid support, wherein the solid support has one or more antibodies immobilized thereon, the one or more antibodies being capable of specifically binding to a polypeptide having an amino acid sequence corresponding to the following amino acid sequence, or an antigenic portion thereof: 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 (D9 peptide), SEQ ID NO: 19 (D10 peptide), SEQ ID NO: 20 (D11 peptide), SEQ ID NO: 21 (D12 peptide), SEQ ID NO: 22 (D13 peptide), SEQ ID NO: 23 (D14 peptide), SEQ ID NO: 24 (D15 peptide), SEQ ID NO: 25 (D16 peptide), SEQ ID NO: 26 (D17 peptide), SEQ ID NO: 27 (D18 peptide), SEQ ID NO: 28 (D19 peptide), SEQ ID NO: 29 (D20 peptide), SEQ ID NO: 30 (D20 peptide), SEQ ID NO: 31 (D20 peptide), SEQ ID NO: 32 (D20 peptide), ID NO:18 (d678 combination), SEQ ID NO:78 (modified d678), SEQ ID NO:79 (d678-amino extension), 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), SEQID 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2 and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2 and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids. The device can be, but is not limited to, for example, an ELISA device, such as a lateral flow immunoassay device or a microtiter plate device. Mammalian samples that can be tested for cat strongyloides by the device include, but are not limited to, for example, blood and related fluids (e.g., serum and plasma); fluids and tissues obtained from pharyngeal swabs, tracheal aspirates, tracheal swabs, bronchoalveolar lavage fluid, pleural effusions, and other materials obtained from the respiratory tract; feces; and whole tissues, such as tissues from the small intestine, large intestine, cecum, colon, rectum, or other tissues obtained from the gastrointestinal tract. The device may also include (but is not required to include) one or more reagents for detecting one or more of the group consisting of: one or more non-cat strongyloides helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, and one or more bacteria. ;

[0013] In another aspect, the present disclosure provides a method for detecting the presence or absence of Strongyloides cati in a sample. The sample can be obtained from a mammal (e.g., a dog, a cat, a pig, a cow, or a human). In one aspect, the method can be performed to test Strongyloides cati antigens in a serum sample. However, the method is not limited to testing serum samples. Therefore, in addition to serum, the sample can also be, for example, but not limited to, blood and related fluids (e.g., plasma); fluids and tissues obtained from pharyngeal swabs, tracheal aspirates, tracheal swabs, bronchoalveolar lavage fluid, pleural effusions, and other materials obtained from the respiratory tract; whole tissues (e.g., tissues from the small intestine, large intestine, cecum, colon, rectum, or other tissues obtained from the gastrointestinal tract). In one embodiment, the steps of the method include contacting the sample with one or more polypeptides of the present disclosure; forming an antibody-polypeptide complex in the presence of antibodies (if any) in the sample; and detecting the presence or absence of the antibody-polypeptide complex (if any). In another embodiment, the steps of the method include contacting the sample with one or more combination polypeptides of the present disclosure; forming an antibody-combination polypeptide complex in the presence of the antibody (if any) in the sample; and detecting the presence or absence of the antibody-combination polypeptide complex (if any). In another embodiment, the steps of the method include contacting the sample with one or more antibodies of the present disclosure; forming an antibody-polypeptide complex in the presence of a cat nematode antigen (if any) in the sample; and detecting the presence or absence of the antibody-polypeptide complex (if any). The method may also include one or more of the following optional steps: diagnosing a mammal as having or not having a cat nematode infection; treating a mammal diagnosed as having a cat nematode infection; and determining the presence or absence of a nucleic acid from cat nematode in the same sample contacted with the polypeptide for detecting the presence or absence of cat nematode or in some other sample from the mammal.

[0014] The method can also be used to test for environmental contamination with conservative variants of one of these sequences. Environmental samples that can be tested by the device include, but are not limited to, soil, rotten matter, or feces or body fluids from residential environments (including courtyards, gardens, sandboxes, amusement parks). Test sites can also include parks, beaches, forests, farms, or other sites exposed to feces or other body materials from other intermediate hosts and transfer hosts of dogs, cats, or cat strongyloides. Feces from indoor and outdoor cat litter boxes can also be tested.

[0015] In another aspect, the present disclosure includes a kit for performing one or more steps of the disclosed method. The kit may optionally include, for example, a device and one or more compositions of the present disclosure and instructions for performing the disclosed method. The kit may also optionally include, for example, one or more indicators, one or more antibody labeling compounds, one or more antibodies, one or more antigen capture reagents, one or more inhibitors, and one or more washing reagents to be used as part of the device and / or for performing the method.

[0016] In another aspect, the present disclosure provides pharmaceutical compositions comprising one or more polypeptides comprising one or more epitopes of a cat nematode antigen or one or more polynucleotides encoding these polypeptides and a physiologically acceptable carrier; and vaccines comprising one or more of the above-mentioned 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 hereby incorporated by reference in their entirety as if each reference were incorporated individually. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows (A) SEQ ID NO: 1 (which is the nucleotide cDNA sequence deduced from Strongyloides cati) and the encoded amino acid sequence SEQ ID NO: 124; (B) shows SEQ ID NO: 20, which is a construct for expressing rTDX1557 (rTDX1557 amino acid sequence is SEQ ID NO: 3); and (C) SDS-PAGE gel analysis of supernatants from mammalian expression systems; and anti-HIS immunoblot analysis as follows: (lane 1) pre-stained marker, (lane 2) recombinant TDX1557 (10 μg / mL), (lane 3) recombinant TDX1557 (5 μg / mL) and (lane 4) recombinant TDX1557 (10 μg / mL). The lanes were probed with mAb HIS labeled with alkaline phosphatase and then developed with BCIP / NBT.

[0019] Figure 2Part (A) shows a comparison of ELISA assays performed using Strongyloides cati lysate and TDX1557 (2M2) with larval shedding (Baermann method) in high shedding cats (BigMac). Part (B) shows a comparison of ELISA assays performed using Strongyloides cati lysate and TDX1557 (2M2) with larval shedding (Baermann method) in low shedding cats (Burrito). The X-axis represents 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. On day 90, cats were treated with antiparasitic drugs. Larval shedding was performed using the standard Baermann method. Serum samples were subjected to ELISA assays using larval lysate and rTDX1557. Results showed that larval shedding was inconsistent, but ELISA assays using lysates and rTDX1557 showed a gradual increase in antibody titers against worms, and antibody titers declined after day 90, indicating that the assays responded to treatment.

[0020] Figure 3 It is shown that no cross-reactivity of anti-TDX1557 (2M2) with heartworm antigens was found. Part A shows SDS-PAGE analysis of cat nematode lysate and heartworm lysate. Part B shows immunoblot analysis of cat nematode lysate and heartworm lysate probed with rabbit pAB against TDX1557 (anti-TDX1557). The rabbit pAB against TDX1557 recognized a single band of ~62 kDa in cat nematode lysate, but did not recognize any bands in heartworm lysate. The positive control was recombinant TDX1557, which also migrated to ~62 kDA.

[0021] Figure 4 Immunoblot analysis of cat nematode lysates and ascaris lysates probed with rabbit pAB against TDX1557 (anti-TDX1557) showed no cross-reactivity of anti-TDX1557 (2M2) with ascaris antigens. Rabbit pAB against TDX1557 recognized a single band of ˜62 kDa in cat nematode lysates, but did not recognize any bands in ascaris lysates. The positive control was recombinant TDX1557, which also migrated to ˜62 kDA.

[0022] Figure 5In part (A) a comparison of high shedding BigMac in fecal samples with ELISA using rTDX1557 and Strongyloides catioides lysate from cat serum samples is shown. Inconsistent shedding rates were observed with fecal samples, whereas antibody responses to rTDX1557 or Strongyloides catioides lysate showed a steady progression. With antiparasitic drugs, antibody responses declined after day 90, indicating that antibodies to rTDX1557 or lysate responded to treatment. Part B shows in tabular form the indirect ELISA for rTDX1557 using serum samples, larval shedding in fecal samples, and BigMac from serum samples collected at different times. Results. Part C shows photographic results of serum samples collected from high shedding BigMac over time (days 0, 7, 14, 28, 63, 91, 98, 126, 168, and 182) and analyzed by SNAP. Part D shows SNAP results of serum samples from field samples of uninfected cats that were also negative for rTDX1557 by indirect ELISA. Of these 10 samples, 9 were found to be negative.

[0023] Figure 6 The amino acid sequences of 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) are shown. These sequences contain an IgG-like signal sequence, a HIS tag marker and an ADX18 epitope tag.

[0024] Figure 7 The predicted secondary structures of the rTDX1557 polypeptide and truncated polypeptides T2, T3, and T4 are shown, as well as SDS-PAGE analysis of the expression of each truncated peptide.

[0025] Figure 8Evaluation of truncated versions of 2M2 (TDX1557) using an indirect ELISA assay is shown. Recombinant polypeptide 2M2 (TDX1557) and truncated polypeptides T2, T3 and T4 were evaluated in an indirect ELISA using rabbit pAbs to TDX1557, temporal cat serum samples (Burrito DPI 98, French Fry DPI 91) and negative cat serum samples (SPF 16CMF3, SPF 16CSL3). Serially diluted serum samples were tested at different concentrations (0.5, 1.5 and 3 μg / ml) of the truncated version of the recombinant. Rabbit pAbs to TDX1557 reacted with all four polypeptides. However, experimentally infected samples did not show any reactivity with the T3 truncated polypeptide, indicating that the important epitope is only present at the carboxyl terminus of the protein. SPF negative controls (SPF 16CMF3 and 16CSL3) showed no reactivity to any antigen. An increase in titration rate was also observed depending on the antigen concentration and dilution rate.

[0026] Fig. 9Evaluation of truncated versions of 2M2 (TDX1557) using an indirect ELISA assay is shown. Recombinant 2M2 as well as truncated T2, T3 and T4 peptides were evaluated in an indirect ELISA using rabbit pAb, temporal (Burrito DPI 98, French Fry DPI 91) and negative samples (SPF 16CMF3, SPF 16CSL3). Serially diluted serum samples were tested at different concentrations (0.5, 1.5 and 3 μg / ml) of the truncated version of the recombinant (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 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 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). Rabbit pAbs against TDX1557 reacted with 2M2 and truncated all four different truncations. However, experimentally infected samples did not show any reactivity with the T3 truncation, indicating that the important epitope is only present at the carboxyl terminus of the protein. SPF negative controls (SPF 16CMF3 and 16CSL3) showed no reactivity to any antigen. An increase in titer was also observed depending on the antigen concentration and dilution rate. ;

[0027] Fig.10Shown are the results of an evaluation of the peptide array using a TDX1557 ELISA-positive naturally infected cat serum sample (8) that showed significant reactivity to peptides D1-D8 and C10-C12. The amino acid sequences of a portion 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; 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: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:7 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).

[0028] Fig.11Shown is an evaluation of the peptide array using experimentally infected cat serum samples that showed positive ELISA assay results using rTDX 1557. The serum samples showed significant reactivity to the peptides. The amino acid sequences of a portion 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; 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: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:7 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).

[0029] Fig.12 Comparison of the immunogenicity of D6, D7, and D8 peptides (SEQ ID NO: 15 for D6–KNLTEMATRASKSWF; SEQ ID NO: 16 for D7-MATRASKSWFDELKK; SEQ ID NO: 17 for D8–SKSWFDELKKFGVPP), Strongyloides cati lysate, and rTDX1557 by ELISA assay using rabbit anti-rTDX1557 pAB is shown. Peptides D6, D7, and D8 were detected by rabbit anti-rTDX1557 pAB at an early stage, indicating their diagnostic utility for infection. ELISA assay using Strongyloides cati larvae lysate and rTDX1557 showed a gradual increase in antibody titers. After treatment with antiparasitic drugs at D90, the antibody titers decreased.

[0030] Fig.13 The secondary structure of the 2M2 polypeptide (SEQ ID NO: 124) predicted using Protter software is shown. Based on the predicted structure, the 2M2 polypeptide is a secreted protein. The amino acid sequence alignment of the immunogenic peptides D6, D7, and D8 (D6 is SEQ ID NO: 15; D7 is SEQ ID NO: 16; D8 is SEQ ID NO: 17) relative to the D678 peptide (CPAQKNLTEMATRASKSWFDELKKFG is SEQ ID NO: 18) is shown, and the overlapping sequences are underlined. The portion of the 2M2 peptide sequence corresponding to the D678 polypeptide is circled.

[0031] Fig.14A comparison of the immunogenicity of D678 polypeptide, Strongyloides felis lysate, and rTDX1557 as determined by ELISA using rabbit anti-rTDX1557 pAB is shown. D678 polypeptide, Strongyloides felis lysate, and rTDX1557 were coated on plates and washed. Cat sera from cats Big Mac, Burrito, chicken Nugget, French Fry, Taco, and Tortilla were then applied to the plates. The plates were washed and rabbit pAB was then applied to the plates. Detection was performed using a secondary antibody. The D678 peptide showed similar reactivity to Strongyloides felis lysate.

[0032] Fig.15 Schematic representation of the extended D678 synthetic peptide sequence showing 2M2, 2M2 amino2Cys, D678 (SEQ ID NO: 78), D678 carboxyl (SEQ ID NO: 80), and D678 amino (SEQ ID NO: 79).

[0033] Fig.16A Shown are the results of an indirect ELISA assay evaluating the immunogenicity of the extended peptide D678, D678 carboxyl, and D678 amino groups using rabbit anti-rTDX1557 pAb and serum samples from temporal and SPF cats.

[0034] Fig. 16B Schematic representation of the truncated 2M2 polypeptides 2M2 amino2Cys, CpepC, T4WO IDR, T4 C2S, T4 1A, T41B and T4 1C and the extended peptides D678, D678 carboxyl and D678 amino are shown.

[0035] Fig.16C1 SEQ ID NO: 2 and the construction of 2M2 full (TDX1557), 2M2T2 (TDX1637), 2M2 T3 (TDX1674), 2M2 T4 (TDX1675), 2M2 amino2cys, 2M2 CpepC and 2M2 Wo epitope and four Cys-Ser variants of 2M2 T4 are shown. The four Cys-Ser variants are 2M2 T4 WO IDR, 2M2 T4 1A, 2M2 T4 1B and 2M2T4 1C.

[0036] Fig.16C2The amino acid sequences of Cys-Ser variants 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) are shown.

[0037] Fig.16D The results of evaluating the immunogenicity of truncations 2M2 amino2cys and 2M2 CpepC (2M2_Amino2Cys_pCDNA34 is SEQ ID NO: 118; 2M2_CpepC_pcDNA34 is SEQ ID NO: 119) by indirect ELISA assay using rabbit anti-rTDX1557 pAb and serum samples from temporal and SPF cats are shown. No reactivity was observed with samples from temporal cats.

[0038] Fig.16E Shown are the results of evaluating the immunogenicity of the truncate 2M2 T4 C2S (2M2_T4C2S is SEQ ID NO: 120) by indirect ELISA assay using rabbit anti-rTDX1557 pAb and serum samples from temporal and SPF cats. No reactivity was observed with samples from temporal cats.

[0039] Fig.17 Shown are immunoblot analyses of Strongyloides cati lysate, rTDX1557 (positive control), and fecal samples from a high larval shedding cat, Big Mac, probed with rabbit pAB to rTDX1557 (anti-TDX1557). Rabbit pAB to rTDX1557 recognized a single band of -62 kDa for Strongyloides cati lysate, rTDX1557, and BigMac fecal samples at days 36, 64, and 141. BigMac was treated with antiparasitic drugs at DPI 90. DETAILED DESCRIPTION Introduction

[0040] The present disclosure generally relates to methods, devices, kits and compositions for detecting Strongyloides cati in samples (e.g., serum) obtained from mammals. In particular, the present disclosure relates to Strongyloides cati polypeptides and conservative variants thereof, polynucleotides encoding these polypeptides and oligonucleotides that specifically bind to these polynucleotides, antibodies generated against these polypeptides and specifically bind to these polypeptides, and methods, devices and kits for detecting Strongyloides cati.

[0041] The present disclosure provides a superior alternative to these existing microscopy techniques. This is because the present disclosure provides compositions, devices, kits, and methods for detecting the presence or absence of Strongyloides cati in samples from mammals that: (1) are both easy to use and produce consistently reliable results; (2) allow confirmation of the presence or absence of Strongyloides cati in mammals, regardless of whether the mammal is infected with hookworms, roundworms, whipworms, heartworms, and / or other feline lung nematodes; and (3) can detect Strongyloides cati before the time when Strongyloides cati larvae first appear in the feces of an infected host.

[0042] The present disclosure is based, in part, on the discovery of unexpected properties of the disclosed compositions. Specifically, it was determined that antibodies of the present disclosure raised against the disclosed polypeptides can be used to capture and detect Strongyloides cati antigens in a mammal even when the mammal is also infected with one or more of hookworms, roundworms, whipworms, heartworms, and / or other feline lung nematodes. This specificity for Strongyloides cati is surprising because whipworms, roundworms, hookworms, heartworms, and other feline lung nematodes are all related nematodes, and antibodies raised against proteins isolated from any of these worms would be expected to cross-react with one or more other worms, host antigens, or other host components.

[0043] It was further determined that the antibody can be used to capture and detect Strongyloides cati antigens in mammals as early as 14 days after the mammals were first infected with Strongyloides cati. This ability to detect Strongyloides cati so soon after infection and before any Strongyloides cati larvae appear in the feces of the infected mammal is surprising because Strongyloides cati larvae do not normally appear in the feces of an infected host until approximately 5-6 weeks after the host was infected.

[0044] Thus, the present disclosure includes methods, devices, compositions and kits for the specific capture and detection of Strongyloides cati antigens in mammals that may also be infected with one or more of roundworms, hookworms, whipworms, heartworms and / or other feline lungworms. The ability of the present disclosure to detect and diagnose Strongyloides cati even in the presence of one or more other worm types allows the caregiver of a mammal the opportunity to optimally select a treatment for clearing Strongyloides cati from the mammal. Furthermore, in some cases, the ability of the present disclosure to detect Strongyloides cati as early as 14 days after the mammal's first infection allows the caregiver to initiate such treatment before the mammal becomes seriously ill from Strongyloides cati. Intervention prior to the appearance of larvae in the feces also greatly reduces or eliminates the possibility of transmission of infection to other animals. Definition and Use of Terms

[0045] The term "composition of the present disclosure" refers to all nucleic acids, polypeptides, antibodies, and mixtures comprising one or more of these nucleic acids, polypeptides, and antibodies and one or more other compounds that are explicitly described, implicitly included, or otherwise disclosed herein and can be used to detect the presence or absence of Strongyloides cati in a sample obtained from a mammal by performing the methods of the present disclosure.

[0046] "Samples from mammals" in which cat roundworms can be detected by the present disclosure include all body components and extracts thereof that can contain cat roundworm antigens, such as any fluid, solid, cell or tissue. Therefore, exemplary samples include, but are not limited to, for example, serum and whole tissues, such as blood and blood components (such as plasma); fluids and tissues obtained from throat swabs, tracheal aspirates, tracheal swabs, bronchoalveolar lavage fluids, pleural effusions and other materials obtained from the respiratory tract; tissues from the small intestine, large intestine, cecum, colon, rectum or other tissues obtained from the gastrointestinal tract. The sample can be obtained directly from the mammal, or the sample can be obtained from any substance that contacts the mammal. For example, the sample can be fresh or rotten feces from the mammal. As another example, the sample can include, for example, soil, dirt, sand, plant material or any other material that can be mixed with body components (such as feces) that can be left by the mammal. Regardless of the source or content of the sample, the sample is sometimes referred to as a "mammalian sample", "test sample" or "sample to be tested" in this article.

[0047] As used herein, "nucleic acid" is synonymous with "gene", "DNA", "cDNA", "EST", "polynucleotide", "oligonucleotide", "polynucleic acid", "RNA" and "mRNA", and can therefore be used interchangeably. The nucleic acid can be in double-stranded form, or can be in single-stranded form. In addition, the nucleic acid can be isolated from nature, such as from the whole cat roundworm or a part thereof; or can be artificially synthesized, whether in a recombinant host organism or by any other artificial method known to those skilled in the art, such as by using PCR-based techniques, by generating transgenic organisms that synthesize the nucleic acid, by using a DNA synthesis machine, or by any other molecular-based technology.

[0048] "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 isolated from nature, e.g., from the whole cat nematode or from a part of the cat nematode; or may be artificially synthesized, whether in a recombinant host organism or by any other artificial method known to those skilled in the art. A polypeptide comprising an epitope may consist entirely of the epitope, or may comprise additional sequences. The additional sequences may be derived from a natural antigen or may be heterologous, and such sequences may (but need not be) antigenic.

[0049] The term "antibody" or "antibody of the disclosure" refers to any antibody that is capable of specifically binding to one or more Strongyloides cati antigens, but does not bind to any antigens from hookworms, roundworms, whipworms, or heartworms. The antibodies of the disclosure may be raised against one or more immunogenic polypeptides of the disclosure. Unless otherwise indicated, it is understood that the antibodies of the disclosure may include a mixture of two or more different types of antibodies. For example, the antibody may be a mixture of two types of antibodies, wherein one of the two types specifically binds to a particular antigen, and the other of the two types specifically binds to some other antigen.

[0050] "Immunogenic polypeptides of the present disclosure" and more simply "polypeptides of the present disclosure" are immunogens against which antibodies of the present disclosure can be produced and / or antibodies that specifically bind to Strongyloides felis present in a sample. All "polypeptides of the present disclosure" are immunogenic and therefore can be used to elicit an immune response in a host animal to produce antibodies of the present disclosure. Unless otherwise indicated, it is understood that a polypeptide of the present disclosure can be one component of a mixed composition of multiple components.

[0051] An "immunogen" is any agent, such as an immunogenic polypeptide of the disclosure, capable of eliciting an immune response in an animal exposed to the agent.

[0052] The term "cat nematode" as used herein refers to nematodes, such as the lung nematodes of the order Rhabditida. Therefore, exemplary lung nematodes include Dictyocaulus viviparus, Angiostrongylus cantonensis and Angiostrongylus vasorum. In addition, the term "lung nematode" as used herein does not refer to the whole of the phylum Nematoda. For example, "lung nematode" does not include any member of Ancylostoma, Uncinaria, Necator, Toxocara, Toxascaris, Ascaris or Dirofilaria.

[0053] A "lungworm antigen" or "antigen of a lungworm" is any Strongyloides felis product that is present in the serum, blood, material from the respiratory tract, or feces of a mammal with a lungworm infection and that can specifically bind to one or more antibodies of the present disclosure. For example, a lungworm antigen can be, but is not limited to, one or more polypeptides of the present disclosure.

[0054] "Lungworm antibodies" or "antibodies to lungworms" are any lungworm antibodies present in the serum or blood of a mammal with a Strongyloides felis infection and that can specifically bind to one or more polypeptides of the present disclosure. For example, a lungworm antibody can be, but is not limited to, an antibody that binds to one or more polypeptides of the present disclosure.

[0055] "Specific for," "specifically binds," and "stably binds" means that a particular composition of the disclosure (e.g., an antibody, polypeptide, or oligonucleotide of the disclosure) recognizes and binds to one or more other agents with a higher affinity than the binding of at least one other agent. As an example, an antibody of the disclosure is said to be "specific for," "specifically binds," and "stably binds" to lungworm antigens when it is able to recognize and bind to those lungworm antigens with a higher affinity than the binding of any other antigen from a non-lungworm parasitic helminth. Such binding specificity can be tested using methods well known in the art, such as ELISA or radioimmunoassay (RIA). Based on the observed information about the binding specificity of a particular composition of the disclosure, the methods of the disclosure can be performed under conditions that allow the composition to bind (and therefore allow detection of such binding) to one or more specific agents, but not to significantly bind to other agents, while maintaining these conditions. As an example, the methods of the disclosure can be performed under conditions that allow the antibodies of the disclosure to bind (and therefore allow detection of such binding) to one or more lungworm antigens present in a particular sample, but not to significantly bind to any hookworm, roundworm, whipworm, heartworm antigen, or other lungworm antigen that may be present in the sample.

[0056] As another example, a polypeptide of the present disclosure is said to be "specific for," "specifically binds," and "stably binds" a lungworm antibody when the antibody is able to recognize and bind with a higher affinity to those lungworm antigenic polypeptides of the present disclosure than to any other antigen from a non-lungworm parasitic helminth. Such binding specificity can be tested using methods well known in the art, such as ELISA or radioimmunoassay (RIA). Based on the information observed about the binding specificity of a particular composition of the present disclosure, the methods of the present disclosure can be performed under conditions that allow the composition to bind (and therefore allow detection of such binding) to one or more specific agents, but not to significantly bind to other agents, while maintaining these conditions. As an example, the methods of the present disclosure can be performed under conditions that allow the antigenic polypeptides of the present disclosure to bind (and therefore allow detection of such binding) to one or more lungworm antibodies present in a particular sample, but not to significantly bind to any hookworm, roundworm, whipworm, heartworm antigens or other non-cat strongyloid lungworms that may be present in the sample.

[0057] "Detecting lungworms" refers to detecting one or more lungworm-specific products, such as Strongyloides cati, including one or more of the polypeptides, antibodies, and nucleic acids of the present disclosure, or one or more lungworm antigens or lungworm antibodies. The presence of one or more such lungworm products in a sample from a mammal indicates that the mammal has Strongyloides cati infection, regardless of whether any whole lungworm organisms or eggs thereof are also present in the sample. Conversely, the absence of one or more such lungworm products in a sample from a mammal indicates that the mammal does not have Strongyloides cati infection.

[0058] "Amino acid" refers to naturally occurring amino acids and synthetic amino acids. Amino acid residues are abbreviated as follows: A or Ala for alanine; R or Arg for arginine; N or Asn for asparagine; D or Asp for aspartic acid; C or Cys for cysteine; E or Glu for glutamic acid; Q or Gln for glutamine; G or Gly for glycine; H or His for histidine; I or Ile for isoleucine; L or Leu for leucine; K or Lys for lysine; M or Met for methionine; F or Phe for phenylalanine; P or Pro for proline; S or Ser for serine; T or Thr for threonine; W or Trp for tryptophan; Y or Tyr for tyrosine; and V or Val for valine. Unless otherwise defined herein, X or Xaa represents any amino acid. Other relevant 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 format, wherein the left-most or first amino acid residue of the sequence is the N-terminal residue and the right-most or last amino acid residue of the sequence is the C-terminal residue.

[0059] "Conservative variants" of any specific nucleic acid sequence include any sequence having one or more degenerate codon substitutions for the specific nucleic acid sequence, any sequence having one or more nucleotide substitutions, insertions and deletions for the specific nucleic acid sequence, and the complementary sequence of the specific nucleic acid and conservative variants of the complementary sequence. Conservative variants of a specific nucleic acid sequence preferably have at least about 85% identity with the specific nucleic acid sequence, more preferably at least about 90% identity, and even more preferably at least about 95-99% identity. Conservative variants of a specific nucleic acid sequence can be artificially synthesized, or it can be isolated from an organism in its natural form, including, for example, isolation from a lung nematode organism (e.g., Strongyloides cati).

[0060] A "conservative variant" of any particular polypeptide sequence is any polypeptide having an amino acid sequence that is different from the amino acid sequence of the particular polypeptide but still retains the specific binding properties of the particular polypeptide, so that the antibodies of the present disclosure produced for the particular polypeptide can specifically bind to the variant polypeptide. Thus, for example, a conservative variant of a particular polypeptide may have one or more amino acid substitutions, deletions, additions and insertions to the particular polypeptide. For example, a conservative variant of a particular polypeptide may have less than 30, less than 25, less than 20, less than 15, less than 10 or less than 5 conservative amino acid substitutions to the particular polypeptide. A conservative variant of a particular polypeptide preferably, but not necessarily, has at least about 80% identity with the particular polypeptide, more preferably has at least about 90% identity, and even more preferably has at least about 91-99% identity. The percentage identity of any subject nucleic acid or amino acid sequence (e.g., any polypeptide described herein) relative to another "target" nucleic acid or amino acid sequence can be determined as follows. First, the target nucleic acid or amino acid sequence of the present disclosure can be compared and aligned with the subject nucleic acid or amino acid sequence using the BLAST 2 Sequences (Bl2seq) program from a stand-alone version of BLASTZ (e.g., version 2.0.14) that includes BLASTN and BLASTP. The stand-alone version of BLASTZ can be obtained at www.ncbi.nlm.nih.gov. Instructions explaining how to use BLASTZ, particularly 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.

[0061] Bl2seq uses BLASTN (for comparing nucleic acid sequences) or BLASTP (for comparing amino acid sequences) algorithm to compare between subject sequence and target sequence.Usually, when performing amino acid sequence alignment, the default parameters of BLOSUM62 scoring matrix are used, the gap existence cost is 11 and the extension cost is 1, the word length is 3, the expected value is 10, the cost per residue is 1 and the lambda ratio is 0.85.The output file contains the alignment region of homology between target sequence and subject sequence.After alignment, the length is determined by calculating the number of continuous nucleotides or amino acid residues (that is, not including gaps) from the target sequence that are aligned with the sequence from the subject sequence starting from any matching position and ending at any other matching position.The matching position is any position where the same nucleotides or amino acid residues are present in both the target sequence and the subject sequence.The gap of one or more residues can be inserted into the target sequence or subject sequence to maximize the sequence alignment between structurally conserved domains (e.g., α helix, β folding and ring).

[0062] The percent identity over a particular length is determined by counting the number of matching positions over the particular length, dividing that number by the length, and multiplying the resulting value by 100. For example, if (i) a 500 amino acid target sequence is compared to a subject amino acid sequence; (ii) the Bl2seq program presents 200 amino acids from the target sequence that are aligned with a region of the subject sequence, wherein the first and last amino acids of the 200 amino acid region are matched; and (iii) the number of matches over the 200 aligned amino acids is 180, then the 500 amino acid target sequence comprises a length of 200, and the sequence identity over that length is 90% (i.e., 180 / 200×100=90). It should be understood that a nucleic acid or amino acid target sequence aligned with a subject sequence can produce many different lengths, each with its own percent identity. It is noteworthy that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. Also note that the length value is always an integer.

[0063] Conservative variants of a particular polypeptide sequence may be artificially synthesized or may be isolated from an organism in its natural form, including, for example, isolation from a lungworm organism (e.g., Strongyloides cati). Those skilled in the art will also recognize that these variants include, but are not limited to, those having replacements of one or more basic amino acid residues, replacements of one or more acidic amino acid residues, replacements of one or more polar amino acid residues, replacements of one or more hydrophobic amino acid residues, replacements of one or more aromatic amino acid residues, and replacements of one or more small amino acid residues. ("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.)

[0064] As used herein, an "epitope" is a portion of an antigenic polypeptide that reacts with serum from a mammal infected with cat strongyloides (i.e., the epitope specifically binds to one or more antibodies in such serum). Epitopes of the polypeptides described in this application can generally be identified using methods known to those of ordinary skill in the art, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. For example, polypeptides derived from natural lungworm antigens produced by recombinant or chemical synthesis methods can be screened for their ability to react with mixed serum obtained from mammals infected with cat strongyloides. Suitable assays for assessing reactivity with lungworm infection serum, such as enzyme-linked immunosorbent assays (ELISA), 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 that of the full-length polypeptide. In other words, the epitope can produce at least about 80%, preferably at least about 100%, of the response produced by the full-length polypeptide in an antibody binding assay (eg, ELISA).

[0065] Combination polypeptides comprising epitopes of multiple antigenic polypeptides are disclosed. A "combination polypeptide" is a polypeptide in which the epitopes of different polypeptides or variants thereof are connected into a single amino acid chain by, for example, a peptide bond. The amino acid chain thus formed can be linear or branched. The epitope can be directly connected (i.e., without intervening amino acids), or can be connected by a linker sequence (e.g., Gly-Cys-Gly) that does not significantly change the antigenic properties of the epitope. The polypeptide epitope can also be connected by a non-peptide bond, such as a heterobifunctional or homobifunctional agent, which is chemically or photochemically coupled, for example, between specific functional groups on the polypeptide epitope by an amino, carboxyl or sulfhydryl group. Bifunctional agents that can be effectively used in the combination polypeptides of the present disclosure are well known to those skilled in the art. The epitope can also be connected by a complementary ligand / anti-ligand pair (e.g., avidin / biotin), wherein one or more epitopes are connected to the first member of the ligand / anti-ligand pair, and then bound to the complementary member of the ligand / anti-ligand pair in solution or in a solid phase. The combination polypeptides may comprise multiple epitopes of the polypeptides described herein and / or may comprise epitopes of one or more other lungworm antigens. Nucleic acids and polypeptides disclosed herein In an attempt to identify a composition that could be used to confirm the presence or absence of lung nematodes in a mammalian sample, a cDNA library of lambda ZapII was constructed using RNA from L1 larvae of Strongyloides felis. The library was screened with serum samples of experimentally infected cats, and several positive clones were identified. These positive clones were cut into 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 cDNA sequence of 1020 nucleotides was deduced from Strongyloides felis (SEQ ID NO: 1) (the sequence is shown in Figure 1A , and identified herein as SEQ ID NO: 1). A BLAST search performed using SEQ ID NO: 1 indicated that a portion of the sequence was similar, but not identical or substantially identical, to a nucleic acid sequence predicted to encode a portion of venom allergen / hookworm secretory protein-like 10. ATG is the start codon of the nucleotide sequence, and TAG is the stop codon of the nucleotide sequence. According to the Signal IP program, the first 60 nucleotide sequence (encoding 20 amino acids) represents the native signal sequence of a secretory protein of Strongyloides felis.

[0066] Analysis of the sequence corresponding to SEQ ID NO: 1 showed that the sequence contained a large ORF. Specifically, Figure 1B As shown, the large ORF of SEQ ID NO: 1 corresponds to nucleotides 1004 to 1960 of SEQ ID NO: 1 and is predicted to encode a polypeptide having the following amino acid sequence: The polypeptides of the present disclosure may be encoded by a nucleic acid having a nucleotide sequence corresponding to all or part of SEQ ID NO: 1 or to all or part of any conservative variants of these sequences. Therefore, it is understood that the amino acid sequence of the polypeptides of the present disclosure is variable.

[0067] For example, a polypeptide of the present disclosure may have an amino acid sequence corresponding to all or part of SEQ ID NO:2, or to all or part of any conservative variant of SEQ ID NO:2.

[0068] In one embodiment, the polypeptide of the present disclosure identified as TDX1557 has the following amino acid sequence:

[0069] The 319 amino acid residues located between the IgG-like signal sequence starting from the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:3 and the HIS tag starting from amino acid residue 341 represent amino acid residues 1-319 of SEQ ID NO:2. As described in the Examples section included herein, the IgG-like signal sequence starting from the N-terminal methionine added to the N-terminus of the polypeptide of SEQ ID NO:2 and 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 techniques. Also as described throughout the Examples section, antibodies raised against the polypeptide corresponding to SEQ ID NO:3 can be used to detect Strongyloides felis antigens. The polypeptide of SEQ ID NO:3 can be interchangeably referred to as 2M2, TDX1557, recombinant TDX1557, or rTDX1557.

[0070] In another specific example, a truncated version of the polypeptide of SEQ ID NO: 2 of the present disclosure is provided. A schematic diagram of the truncated version of the polypeptide of SEQ ID NO: 2 is shown in Fig. 16B In one specific example, the truncated version of the polypeptide of SEQ ID NO: 2 has the following sequence:

[0071] The 197 amino acid residues located between the IgG-like signal sequence starting from the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:4 and the HIS tag starting from 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 included herein, the IgG-like signal sequence starting from the N-terminal methionine added to the N-terminus of the fragment of the polypeptide of SEQ ID NO:2 and the HIS tag and epitope tag added to the C-terminus of the portion of the polypeptide of SEQ ID NO:2 are artificially added by performing standard cloning techniques. Also as described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:4 can be used to detect lungworm antibodies present in samples (e.g., serum) collected from mammals infected with cat roundworms. The polypeptide of SEQ ID NO:4 can be interchangeably referred to as 2M2T2, TDX1637 or T2 polypeptide.

[0072] In another embodiment, the truncated version of the polypeptide of SEQ ID NO: 2 has the following sequence:

[0073] The 196 amino acids located between the IgG-like signal sequence starting from the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:5 and the HIS tag starting from 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 included herein, the IgG-like signal sequence starting from the N-terminal methionine added to the N-terminus of the portion of the polypeptide of SEQ ID NO:2 and the HIS tag and epitope tag added to the C-terminus of the portion of the polypeptide of SEQ ID NO:2 are artificially added by performing standard cloning techniques. Also as described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:5 can be used to detect lungworm antibodies present in samples (e.g., serum) collected from mammals infected with cat roundworms. The polypeptide of SEQ ID NO:5 can be interchangeably referred to as 2M2T3, TDX1674, or T3 polypeptides.

[0074] In one embodiment, the truncated version of the polypeptide of SEQ ID NO: 2 has the following sequence:

[0075] The 130 amino acid residues located between the IgG-like signal sequence starting from the N-terminal methionine residue of the polypeptide corresponding to SEQ ID NO:6 and the HIS tag starting from 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 included herein, the IgG-like signal sequence starting from the N-terminal methionine added to the N-terminus of this portion of the polypeptide of SEQ ID NO:2 and the HIS tag and epitope tag added to the C-terminus of this portion of the polypeptide of SEQ ID NO:2 are artificially added by performing standard cloning techniques. Also as described throughout the Examples section, the polypeptide corresponding to SEQ ID NO:6 can be used to detect lungworm antibodies present in samples (e.g., serum) collected from mammals infected with cat roundworms. The polypeptide of SEQ ID NO:6 can be interchangeably referred to as 2M2_T4, TDX1675, or T4 polypeptide.

[0076] In another specific example, the polypeptide of the present disclosure may 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 specific example, the peptide has the following amino acid sequence:

[0077] C10 MVYDCNVEKTAMIHA(SEQ ID NO:7)

[0078] C11 NVEKTAMIHAKKCVF(SEQ ID NO:8)

[0079] C12 AMIHAKKCVFEHSKG(SEQ ID NO:9)

[0080] D01 KKCVFEHSKGRKDTG(SEQ ID NO:10)

[0081] D02 EHSKGRKDTGENVWV(SEQ ID NO:11)

[0082] D03 RKDTGENVWVMWPAQ(SEQ ID NO:12)

[0083] D04 ENVWVMWPAQKNLTE(SEQ ID NO:13)

[0084] D05MWPAQKNLTEMATRA(SEQ ID NO:14)

[0085] D06 KNLTEMATRASKSWF(SEQ ID NO:15)

[0086] D07 MATRASKSWFDELKK(SEQ ID NO:16)

[0087] D08SKSWFDELKKFGVPP(SEQ ID NO:17)

[0088] These peptides can be used to detect antibodies in a sample, to generate antibodies against lungworm antigens, and / or to design antigenic combination polypeptides for detecting cat roundworm antibodies in a sample. Fig.13 By aligning the polypeptide sequences SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 corresponding to polypeptides D6, D7 and D8 shown in , a combined polypeptide of SEQ ID NO:18 (labeled as D678 or D678 combination) was constructed.

[0089] CPAQKNLTEMATRASKSWFDELKKFG D678 combination (SEQ ID NO: 18)

[0090] It is also contemplated 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 only a portion of a larger polypeptide sequence and may therefore represent, for example, a partial sequence of one or more proteins normally expressed in Strongyloides felis, or 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 appreciate that there are a variety of techniques for artificially fusing two or more polypeptide fragments together.

[0091] It is even further contemplated that the polypeptides of the present disclosure may comprise more than one 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, and SEQ ID NO: 17. Furthermore, it is contemplated that the polypeptides of the present disclosure may comprise a plurality of 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 present disclosure may be formed from a plurality of polypeptide fragments corresponding to SEQ ID NO: 6 fused together. In another example, a polypeptide of the present disclosure can 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.

[0092] Although various polypeptides of the present disclosure are expressed and isolated by specific recombinant techniques or are chemically synthesized (as described in the Examples section included herein), those skilled in the art will recognize that any polypeptide of the present disclosure can be prepared and / or isolated by employing any one or more of a variety of techniques (see, e.g., Sewald and Jakubke, Peptides: Chemistry and Biology, Wiley Publishing (2002); Peptide Synthesis and Applications (Methods in Molecular Biology) Howl, ed., 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 can 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 also include techniques that can be performed to artificially generate 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 can be produced, for example, by employing any one or more of the mutagenesis techniques or by direct synthesis.

[0093] The polypeptides of the present disclosure are useful as reagents for detecting the presence of anti-Streptococcus aureus antibodies in a sample. When immobilized on a substrate and contacted with a sample obtained from a mammal, the immobilized polypeptide is capable of specifically binding to the Strongyloides aureus antibodies, but is not capable of specifically binding to any antibodies from hookworms, roundworms, or heartworms that may be present in the sample. The polypeptides of the present disclosure are suitable for use in capturing only one or more lungworm antibodies, detecting only one or more lungworm antibodies; or more preferably, capturing and detecting one or more lungworm antibodies.

[0094] The polypeptides are also capable of eliciting an immune response in a host animal exposed to the polypeptides to produce one or more antibodies of the present disclosure. Regardless of the technique by which the polypeptides are obtained, when the polypeptides of the present disclosure are to be used as reagents for producing antibodies, the polypeptides of the present disclosure are preferably prepared in a substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90-95% pure, and even more preferably at least about 99% pure. Exemplary techniques for eliciting an immune response in a host organism and for isolating antibodies therefrom are described herein, but it should be understood that the present disclosure is not limited to these techniques. Those skilled in the art will recognize that there are a variety of techniques that can be used to achieve the same goal without departing from the scope and spirit of the present disclosure. Antibodies of the Disclosure

[0095] The present disclosure also includes antibodies and antigen-binding fragments thereof generated against one or more polypeptides of the present disclosure and specifically binding to all or part of the polypeptides, and also includes compositions comprising the antibodies and antigen-binding fragments thereof. When contacted with a sample obtained from a mammal, these antibodies and antigen-binding fragments are able to specifically bind to cat nematode antigens present in the sample, but cannot specifically bind to any antigens from hookworms, roundworms or heartworms that may be present in the sample. The antibodies of the present disclosure are suitable for capturing only one or more lung nematode antigens, for detecting only one or more lung nematode antigens; or more preferably, for capturing and detecting one or more lung nematode antigens.

[0096] Antibodies of the present disclosure can belong to any antibody class, including, for example, IgG, IgM, IgA, IgD, and IgE, and can be prepared by any of a 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 Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Methods Cell. Biol. 37:389-99 (1994); 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), each of which is incorporated herein by reference in its entirety).

[0097] In one technique, for example, a polypeptide of the present disclosure is introduced into a host animal, such as a rabbit, mouse, rat, guinea pig, goat, pig, cattle, sheep, donkey, dog, cat, chicken or horse. An enhanced immune response can be elicited in a host animal by associating the polypeptide with a carrier and / or by exposing the host to an adjuvant, but it should be 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 adjuvant or incomplete adjuvant and MDL-TDM adjuvant. Whether or not the polypeptide is associated with such a carrier or the host is exposed to an adjuvant, booster immunizations can be optionally performed, followed by one or more blood draws from the host animal. Polyclonal antibodies that specifically bind to the polypeptide can then be purified from the antiserum obtained from the one or more blood draws. For example, such purification can be achieved by using affinity chromatography techniques involving associating the polypeptide with a solid support. Such affinity chromatography techniques are well known to those skilled in the art.

[0098] In one embodiment, the antibody of the present disclosure is an antibody produced in a rabbit by immunizing the 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"). The Examples section included herein describes specific techniques for producing and isolating anti-TDX1557 pAB, but those 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.

[0099] In another embodiment, the antibodies of the present disclosure are produced 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, the antibodies of the present disclosure are produced in a host against any one or more polypeptides having an amino acid sequence corresponding to one or more of the following sequences,or one or more polypeptides having an amino acid sequence that is a conservative variant of any of these 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (D678 - amino extension), SEQ ID NO: 80 (D678 - amino extension), SEQ ID NO: 81 (D678 - amino extension), SEQ ID NO: 82 (D678 - amino extension), SEQ ID NO: 83 (D678 - amino extension), SEQ ID NO: 84 (D678 - amino extension), SEQ ID NO: 85 (D678 - amino extension), SEQ ID NO: 86 (D678 - amino extension), SEQ ID NO: 87 (D678 - amino extension), SEQ ID NO: 88 (D678 - amino extension), SEQ ID NO: 89 (D678 - amino extension), SEQ ID NO: 90 (D678 - amino extension), SEQ ID NO: 91 (D678 - amino extension), SEQ ID NO: 92 (D678 - amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F at positions 4 and 5, respectively, are retained,and replacing at least one X at positions 1, 2 and 3 with a conservative or non-conservative amino acid; (b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2 and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids. ,

[0100] In another embodiment, an antibody of the present disclosure is an antibody that specifically binds to one or more polypeptides having an amino acid sequence corresponding to the following sequences, or an antigenic portion thereof: 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D778), SEQ ID NO: 80 (modified D778), SEQ ID NO: 81 (modified D778), SEQ ID NO: 82 (modified D778), SEQ ID NO: 83 (modified D778), SEQ ID NO: 84 (modified D778), SEQ ID NO: 85 (modified D778), SEQ ID NO: 86 (modified D778), SEQ ID NO: 87 (modified D778), SEQ ID NO: 88 (modified D778), SEQ ID NO: 89 (modified D778), SEQ ID NO: 90 (modified D778), SEQ ID NO: 91 (modified D778), SEQ ID NO: 92 (modified D NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2, and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0101] In yet other embodiments, the antibodies of the present disclosure specifically bind to one or more polypeptides having an amino acid sequence that is a conservative variant of a sequence corresponding to 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D778), SEQ ID NO: 80 (modified D778), SEQ ID NO: 81 (modified D778), SEQ ID NO: 82 (modified D778), SEQ ID NO: 83 (modified D778), SEQ ID NO: 84 (modified D778), SEQ ID NO: 85 (modified D778), SEQ ID NO: 86 (modified D778), SEQ ID NO: 87 (modified D778), SEQ ID NO: 88 (modified D778), SEQ ID NO: 89 (modified D778), SEQ ID NO: 90 (modified D778), SEQ ID NO: 91 (modified D778), SEQ ID NO: 92 (modified D778), SEQ ID NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2, and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0102] It should also be understood that the antibodies of the present disclosure can be polyclonal or monoclonal antibodies, single-chain antibodies (scFv), chimeric antibodies and fragments thereof. Monoclonal antibodies specific to the target polypeptide can be obtained and purified by, for example, preparing a cell line that produces antibodies with the desired specificity for the target polypeptide. Such a cell line can be derived from a specific type of cell (e.g., spleen cell) isolated from a host animal that has been previously immunized with the polypeptides described above. In this case, the cells can then be immortalized by, for example, performing any of a variety of fusion techniques known to those skilled in the art by fusing these cells with myeloma cells. In an exemplary technique, cells from an immune host animal are incubated with their fusion partner (e.g., myeloma cells) in the presence of a detergent for a short period of time and then plated on a medium that supports the growth of hybrid cells (but not myeloma fusion partners). This selection can be achieved, for example, by using hypoxanthine, aminopterin, and thymidine (HAT). When hybrid cells appear during the selection process, about one or two weeks after the start of the selection process, the ability of individual hybrid colonies (and their supernatants) to bind to one or more polypeptides with which the host animal is immunized is tested. The hybridizing colonies with the best binding specificity will represent the best candidates from which monoclonal antibodies can be isolated. For example, these monoclonal antibodies can be isolated directly from the supernatant (i.e., culture medium) in which these colonies are grown by employing any of a variety of techniques known to those skilled in the art.

[0103] The antibodies of the present disclosure may also be single-chain antibodies (scFv) or antigen-binding fragments of antibodies. Antibody antigen-binding fragments are a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, wherein the portion does not contain the constant heavy chain domain of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and F vFragments. In addition to production and purification from animal or mammalian cells, antibodies, antibody fragments or non-antibody scaffolds can also be selected based on various in vitro techniques including phage display, ribosome display or bacterial display.

[0104] Antibodies, including secondary antibodies, can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal particles, radioisotope, and bioluminescent labels. In various embodiments of the present disclosure, one or more antibodies of the present disclosure are labeled with enzymes, colloidal particles, radionuclides, or fluorophores. The particle labels can be, for example, colored latex particles, dye sols, or gold sols conjugated to the antibodies. Methods, devices and kits disclosed herein Devices and kits disclosed herein

[0105] The present disclosure in one aspect is, for example, a device for detecting Strongyloides infectivity in a mammal (e.g., a dog, cat, pig, cattle, or human). The device is arranged to facilitate the detection of Strongyloides antibodies using the polypeptides of the present disclosure (or Strongyloides antigens using the antibodies of the present disclosure) in a sample from a mammal that may also be infected with one or more other helminth parasites (including hookworms, roundworms, whipworms, and heartworms).

[0106] In one aspect, the device comprises a solid support, wherein 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, for example, the inner bottom surface of a well of a microtiter plate or a substrate included as part of a lateral flow device. An exemplary microtiter plate is an Immulon 1B 96-well plate (which is commercially available from Thermo Scientific of Milford, MA), but it should be understood that a variety of other microtiter plates other than the Immulon 1B 96-well plate also allow for the immobilization of polypeptides (or antibodies) thereon, and are therefore suitable for providing a solid support of the present disclosure.

[0107] An exemplary lateral flow device is the lateral flow device described in U.S. Pat. No. 5,726,010, which is incorporated herein by reference in its entirety. A device for performing a lateral flow assay can be device, which is commercially available from IDEXX Laboratories, Inc. of Westbrook, ME. However, it should be understood that those skilled in the art will recognize that it is not A number of other lateral flow devices not described in US Pat. No. 5,726,010 also allow for the immobilization of polypeptides (or antibodies) thereon and are therefore suitable for use as devices of the present disclosure. These devices may include, for example, lateral flow devices using colloidal gold technology.

[0108] The polypeptides or antibodies used in the devices of the present disclosure can be immobilized on the solid support by any method known in the art, including, for example, covalently or non-covalently, directly or indirectly attaching the antibody to the solid support. Thus, although these antibodies or polypeptides can be attached to the solid support by physical adsorption (i.e., without the use of chemical linkers), these antibodies or polypeptides can also be immobilized to the solid support by any chemical binding (i.e., using chemical linkers) method well known to those skilled in the art.

[0109] It is also understood that the solid support can be any suitable material for immobilizing the antibodies or polypeptides of the present disclosure. For example, the solid support can be beads, particles, tubes, wells, probes, test strips, pipette tips, slides, fibers, membranes, paper, natural and modified cellulose, polyacrylamide, agarose, glass, polypropylene, polyethylene, polystyrene, dextran, nylon, amylase, plastic, magnetite, or any other suitable material known to those skilled in the art.

[0110] The device may optionally include one or more labeled antigen or antibody capture reagents, which may be mixed with a sample from a mammal before being applied to the device of the present disclosure. When a labeled antigen or antibody capture reagent is included, the labeled antigen or antibody capture reagent may or may not be deposited or dried on the solid surface of the device. "Antigen capture reagent" refers to any compound that is specific for one or more target antigens. The labeled antigen capture reagent, whether added to the mammalian sample or pre-deposited on the device, can be, for example, a labeled antibody that is specific for a cat nematode antigen, including but not limited to the antibodies of the present disclosure. In one example, anti-TDX1557 pAB conjugated to horseradish peroxidase can be used as a labeled antigen capture reagent. "Antibody capture reagent" refers to any compound that specifically binds to one or more target antibodies. The labeled antibody capture reagent, whether added to the mammalian sample or pre-deposited on the device, can be, for example, a labeled polypeptide that is specific for a cat nematode antibody, including but not limited to the polypeptides of the present disclosure. In one example, the recombinant protein TDX1557 conjugated to horseradish peroxidase can be used as a labeled antibody capture reagent.

[0111] The device may also optionally contain liquid reagents that are transported from the reaction zone (solid phase) (e.g. when the device is The liquid reagent may be a washing reagent and used only to remove unbound materials from the reaction zone; or the liquid reagent may contain a detection reagent and used to remove unbound materials and promote antigen or antibody detection. For example, in the case of an antigen or antibody capture reagent conjugated to an enzyme, the detection reagent includes a substrate that produces a detectable signal when reacting with the enzyme-antibody conjugate in the reaction zone (solid phase). Alternatively, in the case of a labeled antigen or antibody capture reagent conjugated to a radioactive, fluorescent or light-absorbing molecule, the liquid reagent is used only as a washing solution, which promotes the detection of complex formation at the reaction zone by washing away unbound labeled reagents.

[0112] The liquid reagent may also contain a limited amount of an "inhibitor," a substance that blocks the development of a detectable final product. The limited amount is defined as an amount of inhibitor that is sufficient to block the development of the final product until most or all of the excess unbound material is transported away from the second region, at which point a detectable final product is produced.

[0113] The device of the present disclosure may also include various binding reagents fixed at positions different from one or more antigen or antibody capture reagents. For example, an immune reagent (antibody, antigen or polypeptide) of a species-specific (e.g., cat nematode-specific) antibody portion of an antibody or antigen capture reagent that recognizes a label or an enzyme portion of an enzyme-labeled reagent may be included as a positive control to assess the activity of the reagent in the device. For example, a positive control may be an anti-horseradish peroxidase antibody produced in, for example, goats or mice. In addition, a reagent (e.g., an antibody) separated from a non-immune member of the antibody portion of the antigen-antibody complex source species may be included as a negative control to assess the specificity of the immune complex (i.e., antigen-antibody complex) formed.

[0114] In addition to being designed for detecting Strongyloides felis in mammalian samples, the devices of the present disclosure may optionally be designed to allow for the performance of one or more other diagnostic tests. For example, the solid support may also contain reagents for detecting one or more non-Streptostrongyloid lungworm parasites, one or more non-lungworm helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria. The reagents for detecting one or more non-lungworm helminth parasites, 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 that are recognized by antibodies specific for one or more non-lungworm helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria.

[0115] In one embodiment, the device of the disclosure is a microtiter plate comprising a plurality of wells, wherein each well comprises a solid support having immobilized thereon a polypeptide of the disclosure, such as recombinant TDX1557, a conservative variant of TDX1557, or a truncated version of TDX1557.

[0116] The plate can be used in conjunction with the methods of the present disclosure to detect Strongyloides felis lungworms in mammalian samples. Specifically, lungworm infection in mammals can be diagnosed by detecting one or more lungworm antibodies with a polypeptide of the present disclosure (e.g., recombinant TDX1557, a conservative variant of TDX1557, or a truncated version of TDX1557) immobilized on a solid support. In one embodiment, the antibody detected is a lungworm antibody. "Lungworm antibodies" are antibodies directed against any one or more products of lungworms present in a sample (e.g., a serum sample) and capable of specifically and stably binding to recombinant TDX1557, a conservative variant of TDX1557, or a truncated version of TDX1557. Therefore, lungworm antibodies can be antibodies against whole lungworms, lungworm eggs, lungworm fragments, or products secreted, excreted, or shed by lungworms, or a combination thereof. Lungworm antigens, for example, can include any of the polypeptides disclosed herein, such as polypeptides having an amino acid sequence corresponding to SEQ ID NO: 2 to SEQ ID NO: 17, polypeptides having an amino acid sequence that is a conservative variant of these sequences, and / or antigenic fragments of any such polypeptides.

[0117] The present disclosure also includes an assay kit (e.g., a manufactured product) for detecting Strongyloides felis lungworms in mammalian samples. 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 tool for determining the binding of an antigenic polypeptide of the present disclosure to a lungworm antibody in a sample or the binding of an antibody of the present disclosure to a lungworm antigen in a sample. In a specific example, such a kit includes, for example, a device having a fixed polypeptide of the present disclosure (e.g., rTDX1557), one or more antibody capture reagents (e.g., non-fixed labeled antibody capture reagents and fixed antibody capture reagents) and washing reagents, as well as detection reagents and positive and negative control reagents (if necessary or appropriate). Other components known to those of ordinary skill in the art (e.g., buffers, controls, etc.) may be included in such a test kit. The relative amounts of various reagents may vary to provide a concentration of the reagent in solution that significantly optimizes the sensitivity of the assay. Specifically, the reagent may be provided as a dry powder, typically lyophilized, which, after dissolution, will provide a reagent solution having an appropriate concentration to be combined with the sample. The kits may also contain instructions for performing one or more methods of the disclosure, including instructions for using any of the devices and / or compositions of the disclosure contained in the kit. Methods of the present disclosure

[0118] The present disclosure also includes methods for detecting the presence or absence of Strongyloides felis lungworms in a sample using one or more of the devices, kits, and / or compositions of the present disclosure. Thus, the method can be performed to detect the presence or absence of lungworms in a sample (e.g., a serum sample) obtained from a mammal (including but not limited to a dog, cat, pig, cattle, or human). In addition, the method can be performed to detect, for example, roundworms, hookworms, whipworms, and / or heartworms. These methods can also be used to confirm the presence or absence of lungworms in the sample even in the case where the sample contains one or more products from other helminth species (including one or more products from hookworms, whipworms, roundworms, and / or heartworms).

[0119] In one embodiment of the disclosed method, detection of Strongyloides felis lungworms can be accomplished by detecting the presence or absence of one or more lungworm antibodies in a sample using a polypeptide of the disclosed invention (e.g., a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 2 to SEQ ID NO: 18, and fragments and / or conservative variants of these sequences). In another embodiment of the disclosed invention, detection of lungworms can be accomplished by detecting the presence or absence of one or more lungworm antigens in a sample using an antibody of the disclosed invention (e.g., rTDX1557 pAb). One skilled in the art will recognize that there are also a variety of methods for extracting and preparing non-fecal samples from mammals. For example, the sample can be a serum sample obtained from blood or a sample obtained by swabbing a mammal, such as blood and related fluids (e.g., serum and plasma); fluids and tissues obtained from pharyngeal swabs, tracheal aspirates, tracheal swabs, bronchoalveolar lavage fluids, pleural effusions, and other materials obtained from the respiratory tract of a mammal. As another example, tissue sections can be obtained by biopsy, including tissues from the small intestine, large intestine, cecum, colon, rectum, or other tissues of the gastrointestinal tract.

[0120] One embodiment of the method includes contacting a mammalian sample with one or more polypeptides of the present disclosure that are specific for one or more Strongyloides felis lungworm antibodies under conditions that allow the formation of antigen / antibody complexes (i.e., immune complexes). That is, antigenic polypeptides that specifically bind to lungworm antibodies present in the sample. In another embodiment of the method, it includes contacting a mammalian sample with one or more antibodies of the present disclosure that are specific for one or more lungworm antigens under conditions that allow the formation of antigen / antibody complexes (i.e., immune complexes). That is, antibodies that specifically bind to lungworm antigens 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, a second antibody that binds to the antigen / antibody complex can be used to detect the antigen / antibody complex. Any suitable method known in the art can be used to detect the formation of a complex between a polypeptide of the present disclosure and a lungworm antibody in a sample.

[0121] In addition, the relative amount of antibody-antigen complexes formed in a particular reaction relative to the antibody-antigen complexes formed in any other reaction can be measured by any method known in the art for achieving this purpose. When it is determined that the test sample has more antibody-antigen complexes than the control sample, a conclusion that lungworms are present in the test sample can be drawn. When this is true, a conclusion that the mammal from which the test sample was obtained has a lungworm infection can be drawn. For example, one or both of the conclusions that lungworms are present in the test sample and that the tested mammal has a lungworm infection can be drawn by a clinician at a diagnostic service provider or a caregiver of the mammal (e.g., a veterinarian of the mammal). When the caregiver of the mammal determines (or is otherwise informed) that the mammal has a lungworm infection, the caregiver can then optimally design a treatment course for the mammal to specifically eliminate the lungworms of the mammal rather than generally eliminate parasitic nematode infections. In addition, the present disclosure can be used to confirm that any animal that has received treatment for lungworm infection has gotten rid of the infection.

[0122] The steps of the method of the present disclosure may include applying a mammalian sample to a device of the present disclosure, the device comprising an immobilized polypeptide specific for one or more Strongyloides felis lungworm antibodies (or an immobilized antibody specific for one or more lungworm antigens); and detecting the presence or absence of lungworm antibodies (or antigens) in the sample. The polypeptide specific for lungworm antibodies or the antibody specific for lungworm antigens may be attached directly or indirectly to a solid support or substrate, such as a microtiter well, The polypeptide (or antibody) immobilization part of the device, magnetic beads, non-magnetic beads, columns, matrices, membranes, fiber mats composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, such as polyethylene, polypropylene or polyester), sintered structures composed of granular materials (e.g., glass or various thermoplastic polymers), or cast membrane films composed of nitrocellulose, nylon, polysulfone, etc. (usually synthetic in nature). All of these substrate materials can be used in a suitable shape, such as a membrane, sheet or plate, or they can be coated or bonded or laminated to a suitable inert carrier, such as paper, glass, plastic film or fabric. Suitable methods for fixing peptides on a solid phase include ionic, hydrophobic, covalent interactions, etc.

[0123] However, the methods of the present disclosure do not require the use of a solid phase or substrate. Those skilled in the art will recognize that there are a variety of ways in which the present methods can be performed to detect the presence or absence of Strongyloides felis lungworms without involving 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.

[0124] In some embodiments of the present disclosure, the antigen / antibody complex is detected when an indicator (e.g., an enzyme conjugate bound to the antibody) catalyzes a detectable reaction. Optionally, an indicator comprising a signal generating compound can be applied to the antigen / antibody complex under conditions that allow the formation of a detectable antigen / antibody / indicator complex. Optionally, the antibody can be labeled with an indicator before the antigen / antibody complex is formed.

[0125] The formation of antigen / antibody complexes or antigen / antibody / indicator complexes in some methods of the present disclosure can be particularly detected by radioassay, colorimetry, fluorescence, photometry, size separation or precipitation. Detection of antigen / antibody complexes can also be achieved by adding a second antibody coupled to an indicator containing a signal generating compound. Indicators containing signal generating compounds (labels) associated with polypeptide / antibody complexes can be detected using the above methods and can include chromogens, catalysts (e.g., enzyme conjugates), fluorescent compounds (e.g., fluorescein and rhodamine), chemiluminescent compounds (e.g., dioxetane, acridinium, phenanthridinium, ruthenium and luminol), radioactive elements, direct visual markers, and cofactors, inhibitors, magnetic particles, etc. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, β-galactosidase, etc. The choice of a specific label is not important, but it will be able to generate a signal alone or in combination with one or more other substances.

[0126] Methods of the present disclosure include, but are not limited to, methods based on competition, direct reaction, or sandwich-type assays, including, but not limited to, ELISA, RIA, immunofluorescence assay (IFA), hemagglutination reaction (HA), fluorescence polarization immunoassay (FPIA), and microtiter plate assays (i.e., any assay performed in one or more wells of a microtiter plate). One assay of the present disclosure includes a reversible flow chromatography binding assay, which can be performed, for example, by using See U.S. Patent No. 5,726,010.

[0127] In some embodiments, the methods of the present disclosure facilitate sandwich or competitive specific binding assays. In sandwich assays, antibody capture reagents are immobilized in a reaction zone. These antibody capture reagents can specifically bind to antibodies against lungworms in a test sample. After binding of antibodies from the sample, the antibody capture reagent / antibody complex is detected by any suitable method. For example, the complex can react with a labeled specific binding reagent (e.g., an enzyme-antibody conjugate) and detect the antibody (e.g., by reacting with a substrate).

[0128] In other embodiments of the disclosed methods, a competitive assay is performed. In a competitive assay, an antibody capture reagent is fixed to a reaction zone and is simultaneously contacted with antibodies from a sample and labeled antibodies (e.g., antibody-enzyme conjugates). The amount of label detected in the reaction zone is inversely proportional to the amount of antibody in the sample.

[0129] In some embodiments of the method, a polypeptide that specifically binds to one or more Strongyloides felis lungworm antibodies is attached to a solid phase or substrate. A sample that may contain 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 an indicator, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color allowed to develop. The color development reaction may be stopped and the color may be quantified using, for example, a spectrophotometer and / or the color may be assessed subjectively by the human eye.

[0130] In other embodiments of the method, a polypeptide specific for one or more Strongyloides felis lungworm antibodies is attached to a solid phase or substrate. A sample that may contain lungworm antibodies is added to the substrate. Second anti-species antibodies that specifically bind to the lungworm antibodies 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 contain an indicator, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color allowed to develop. The color reaction may be stopped and the color may be quantified using, for example, a spectrophotometer and / or the color may be subjectively assessed by the human eye.

[0131] In a specific example, the method of the present disclosure is implemented in conjunction with the device by adding a prepared mammalian sample to a flow matrix of a device as a lateral flow assay device in a first area (sample application area). The prepared sample is transported to a second area of ​​the flow matrix by capillary action in a fluid flow path, where there are particle markers that can bind to antibodies in the sample and form a first complex. The particle markers can be, for example, colored latex particles, dye sols, or gold sols conjugated to antibodies specific for cat strongyloides lungworm antibodies. The first complex is transported to a third area of ​​the flow matrix, where polypeptides that specifically bind to lungworm antibodies are fixed at different locations. A second complex is formed between the fixed polypeptide and the first complex. The particle markers that are part of the second complex can be directly seen by the human eye.

[0132] The polypeptide can be an immobilized antibody capture reagent in the reaction zone (solid phase). A second antibody capture reagent, i.e., a second antibody conjugated to a label, can be added to the sample before the sample is added to the device, or the second antibody capture reagent can be incorporated into the device. For example, the labeled antibody capture reagent can be deposited and dried in 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 can result in dissolution of the labeled antibody capture reagent.

[0133] In other embodiments of the method, antibodies that specifically bind to one or more Strongyloides felis lungworm antigens are attached to a solid phase or substrate. A sample that may contain one or more antigens from the lungworm is added to the substrate. Second antibodies that specifically bind to the lungworm antigens are added. These second antibodies can be linked to an indicator, such as an enzyme conjugate. A washing step can be performed before each addition. A chromophore or enzyme substrate can be added and color allowed to develop. The color reaction can be stopped and the color can be quantified using, for example, a spectrophotometer and / or the color can be subjectively assessed by the human eye.

[0134] In other embodiments of the method, antibodies specific for one or more Strongyloides felis lungworm antigens are attached to a solid phase or substrate. A sample that may contain lungworm antigens is added to the substrate. Second anti-species antibodies that specifically bind to lungworm antigens 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 contain an indicator, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate may be added and color allowed to develop. The color reaction may be stopped and the color may be quantified using, for example, a spectrophotometer and / or the color may be subjectively assessed by the human eye.

[0135] In a specific example, the method of the present disclosure is implemented in conjunction with the device by adding a prepared mammalian sample to a flow matrix of a device as a lateral flow assay device in a first area (sample application area). The prepared sample is transported to a second area of ​​the flow matrix by capillary action in a fluid flow path, where there are particle markers that can bind to antigens in the sample and form a first complex. The particle markers can be, for example, colored latex particles, dye sols, or gold sols conjugated to antibodies specific for lungworm antigens. The first complex is transported to a third area of ​​the flow matrix, where antibodies that specifically bind to lungworm antigens are fixed at different locations. A second complex is formed between the fixed antibodies and the first complex. The particle markers that are part of the second complex can be directly seen by the human eye.

[0136] The antibody can be an immobilized antigen capture reagent in the reaction zone (solid phase). A second antigen capture reagent, i.e., a second antibody conjugated to a label, can be added to the sample before the sample is added to the device, or the second antigen capture reagent can be incorporated into the device. For example, the labeled antigen capture reagent can be deposited and dried in 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 cause the labeled antigen capture reagent to dissolve.

[0137] In one embodiment of the disclosed method, Strongyloides felis lungworm antibodies (or Strongyloides felis lungworm antigens) are detected by ELISA. Specific examples of the disclosed ELISA methods are described in the Examples section included herein. Although the disclosure has been described with respect to those specific ELISA methods, however, it should be understood that one of ordinary skill in the art will recognize that alternative, additional or alternative ELISA steps may be used without departing from the basic objectives achieved by this method of the disclosure.

[0138] In another embodiment of the disclosed method, for example, by using a lateral flow device (e.g. Device) to detect Strongyloides felis lungworm antibodies (or Strongyloides felis lungworm antigens).

[0139] In addition, the methods 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 assays 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 a combination thereof. By providing two or more unique binding sites (e.g., In one embodiment, there are three unique spots (these spots are antigen or antibody binding reagents) for detecting past or present infection or infestation of three organisms from a single sample (i.e., the same individual sample is exposed to three capture reagents on a single device). In another embodiment, there are four unique spots (these spots are antigen or antibody binding reagents) for detecting past or present infection or infestation of 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.

[0140] Reagents for detecting one or more non-lungworm helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria can be, for example, one or more antibodies or one or more antigens that are recognized by antibodies specific for one or more non-lungworm helminth parasites, one or more non-helminth parasites, one or more viruses, one or more fungi, or one or more bacteria.

[0141] When the device of the present disclosure contains, for example, reagents for specifically detecting hookworms, reagents for specifically detecting lungworms, and reagents for specifically detecting roundworms in addition to reagents for detecting lungworms, the methods of the present disclosure may involve using the device for one or more additional purposes of determining whether a sample being tested for lungworms also contains hookworms, whipworms, roundworms, and / or heartworms. Thus, in this arrangement, the method / device of the present disclosure is not only able to specifically confirm the presence or absence of lungworms in any particular test sample, it can also be used to specifically confirm that the sample contains or does not contain any hookworm antigens, any whipworm antigens, and / or any roundworm antigens. The ability to specifically detect lungworms and one or more other organisms by applying a single sample to the device of the present disclosure is very useful to caregivers of animals from which test samples are obtained. For example, a caregiver who knows that a sample contains both lungworms and roundworms but not whipworms or hookworms can use this knowledge to treat the mammal from which the sample was collected specifically for lungworms by giving the mammal a drug that is most effective for lungworms and a second drug that is most effective for roundworms. Without such knowledge, the caregiver might, for example, treat the mammal with a drug that is only most effective for lungworms, only most effective for roundworms, or not most effective for both lungworms and roundworms (in which case the mammal would be at risk of receiving suboptimal treatment). In addition, humans who may come into contact with infected animals or their excreta may be advised to take precautions to avoid infection with parasites. In this case, it is very important to determine the worm species with high specificity because some worms (such as roundworms and hookworms) can cause serious diseases in humans (i.e., larval migrans, severe enteritis, or allergic reactions), while lungworms are generally believed to not play an important zoonotic role in humans.

[0142] The method may also optionally include using one or more nucleic acids from lungworms (including, but not limited to, nucleic acids of the present disclosure) to determine the presence or absence of lungworms in a mammalian sample. This use of these nucleic acids to determine the presence of lungworms can be performed before, after, or simultaneously with any other aspect of the method (including detection of lungworms by antibodies). Thus, in one aspect, after lungworms are detected or not detected in a particular sample and the mammal from which the sample was obtained is diagnosed as having or not having a lungworm infection, the sample (or a sample subsequently obtained from the diagnosed mammal) can be tested for the presence or absence of any one or more nucleic acids (including any one or more nucleic acids of the present disclosure). When one fails to detect lungworms in a particular mammal by using one or more nucleic acids (after detecting lungworms by using one or more antibodies), the possibility that the antibodies detected lungworm antigens before detectable lungworm nucleic acids appeared in the sample needs to be considered. In this case, the mammal's caregiver can choose to ignore the observation that the nucleic acids failed to detect lungworms and continue to treat the mammal specifically for lungworm infection based on the observation that the antibodies actually detected lungworms. In another aspect, the nucleic acid is used to determine the presence or absence of lung nematodes in a particular mammal, which is then further assessed by using the antibodies disclosed herein. Detection of one or more lung nematode nucleic acids can be performed using any nucleic acid detection technique known to those skilled in the art. For example, such detection can be performed by performing a PCR-based technique, such as, but not limited to, a real-time PCR-based technique. Exemplary PCR-based techniques are described, for example, in PCR Protocols (Methods in Molecular Biology), 2 nd ed., Bartlett and Stirling, eds., Humana Press (2003); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001); each of which is incorporated herein by reference in its entirety. Vaccines and pharmaceutical compositions

[0143] In another aspect of the present disclosure, vaccines and pharmaceutical compositions for preventing Strongyloides cati infection and its complications in mammals are provided. The pharmaceutical composition generally comprises one or more polypeptides containing one or more epitopes of Strongyloides cati protein, or comprises one or more polynucleotides encoding one or more of the polypeptides, and a physiologically acceptable carrier. The vaccine comprises one or more of the above-mentioned polypeptides or polynucleotides, and an adjuvant for enhancing the immune response.

[0144] The polypeptides as defined above and the polynucleotides encoding these polypeptides can be used for the preparation of immunogenic compositions, in particular vaccines against Strongyloides felis.

[0145] The immunogenic composition comprising one or more polypeptides as defined above or one or more polynucleotides encoding the polypeptides may be combined with one or more adjuvants for enhancing the immune response. In some embodiments, the composition may comprise one or more other immunogenic polypeptides recognized by anti-Streptostrongylus felis antibodies.

[0146] In some embodiments, an immunogenic composition according to the present disclosure is a vaccine.

[0147] Where appropriate, in particular in the case of short peptides (≤30 amino acids), the polypeptides may be coupled to a carrier protein. As examples of carrier proteins, mention may be made of KLH (keyhole limpethemocyanin), 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 or by means of polymer chains (e.g. polylysine).

[0148] If polynucleotides are used as immunogens, the immunogenic composition can be in the form of a recombinant vector in which the polynucleotides to be administered are inserted. For example, viral vectors such as poxvirus, adenovirus, retrovirus, slow virus, herpes virus and AAV (adeno-associated virus) etc. can be used. It can also be in the form of non-pathogenic bacteria transformed with one or more expression vectors comprising the polynucleotides. The polynucleotides can also be directly administered in the form of naked DNA, or incorporated into liposomes. In the case of vaccines, non-pathogenic bacteria (such as lactobacilli, or non-pathogenic strains of Escherichia coli (Escherichiacoli) or Salmonella suis) or vectors derived from vaccine virus strains may be useful.

[0149] In one embodiment, the polynucleotides of the present disclosure can be administered to a mammal in a manner such that the molecule can be expressed as a protective protein (e.g., mRNA) or protective RNA (e.g., antisense RNA, ribozyme, or RNA drug) in a mammal to be protected from disease. Nucleic acid molecules can be delivered to mammals in a variety of ways, including but not limited to (a) direct injection (e.g., as "naked" DNA or RNA molecules, such as taught in Wolff et al., 1990, Science 247, 1465-1468); (b) packaged as a recombinant viral particle vaccine or a recombinant cell vaccine (i.e., delivered to cells by a vector selected from the group consisting of a recombinant viral particle vaccine and a recombinant cell vaccine); or (c) encapsulated in a liposome or lipid nanoparticle. In some embodiments, the polynucleotide may contain one or more modified uridine residues, such as pseudouridine or methyl pseudouridine.

[0150] The recombinant viral particle vaccine of the present disclosure comprises a recombinant molecule of the present disclosure, which is packaged in a viral coat and can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging-defective. A variety of recombinant viral particles can be used, including but not limited to recombinant viral particles based on alphavirus, poxvirus, adenovirus, herpes virus and retrovirus. Methods for producing and using recombinant viral particle vaccines are known in the art.

[0151] Any of a variety of adjuvants can be used in the immunogenic compositions of the present disclosure to non-specifically enhance the immune response, for example by increasing the immunogenicity of the polypeptide. Most adjuvants contain substances designed to protect the antigen from rapid catabolism (e.g., alum (aluminum hydroxide) or mineral oil) and non-specific immune response stimulants (e.g., lipid A, Bordetella pertussis, or Mycobacterium tuberculosis). Such adjuvants are commercially available, such as Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), liposomes, virosomes (reconstructing the viral envelope), peptide derivatives of muramic acid, etc. In the case of vaccines, a pharmacologically acceptable adjuvant will certainly be selected; as an example of an adjuvant, an adjuvant of the "oil-in-water" emulsion type will be mentioned.

[0152] Although any suitable carrier known to those of ordinary skill in the art can be used for pharmaceutical compositions of the present disclosure, the carrier type will vary according to the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably includes water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above-mentioned carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum powder, cellulose, glucose, sucrose and magnesium carbonate. Biodegradable microspheres (such as polylactic acid galactoside) can also be used as carriers of pharmaceutical compositions of the present disclosure.

[0153] The route and frequency of administration and the polypeptide or polynucleotide dosage will vary from individual to individual, and can be similar to the route and frequency currently used for immunization against other protozoan infections. In general, pharmaceutical compositions and vaccines can be administered by injection (e.g., intramuscularly, intravenously or subcutaneously), intranasally (e.g., by inhalation) or orally. 1 to 4 doses can be administered within 2-6 weeks. In one embodiment, two doses are administered, wherein the second dose is 2-4 weeks later than the first dose. Suitable dosage is the amount of polypeptides that effectively produces antibodies sufficient to protect mammals from cat roundworm infection within a period of time in the treated mammal. In general, the scope of the amount of polypeptides present in the dosage is about 1 μg to about 100 mg per kilogram of host, usually about 10 μg to about 1 mg, preferably about 100 μg to about 1 μg. Suitable dosage size will vary with the size of the animal, but for 10-60 kg animals, it is usually in the range of about 0.01 mL to about 5 mL.

[0154] The present disclosure is described in detail with reference to certain specific embodiments; however, it should not be construed as being limited thereto. Example

[0155] Unless otherwise noted, the following materials and techniques were used to generate the data described in one or more of Examples 1-12 described below.

[0156] Preparation of Feline Lungworm Extract Strongyloides felis worms were obtained from Dr. Bowman at Cornell University, Ithaca, NY. Whole worms were washed several times with cold PBS, pH 7.0, to remove any fecal material and mucus from the host at room temperature and homogenized with an ultrasonicator at 4°C until no tissue clumps were visible to the naked eye. The homogenized material was centrifuged at 10,000 g for 30 minutes at 4°C and the supernatant was carefully removed. Protein concentration was determined using the Bradford assay.

[0157] Heartworm Extract PreparationHeartworms [Dirofilaria immitis] were obtained from IDEXX Laboratories, Westbrook, ME. Whole worms were broken and suspended in cold PBS, pH 7.0, and homogenized at 4°C using a polytron homogenizer. 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 3. Protein concentration was determined using the Bradford assay.

[0158] Ascaris extract preparation. Ascaris [Toxocara spp.] were obtained from Antibody systems Inc, Hurst, TX, and processed as described in U.S. Patent No. 7,951,547 (incorporated by reference in its entirety). Whole worms were broken up and suspended in cold PBS, pH 7.0, and homogenized at 4°C using a polytron homogenizer [Kinematica, Bohemia, NY]. 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.

[0159] Larval shedding procedure : To detect lungworm in cats, a visual fecal Baermann test is usually used to detect parasite larvae rather than eggs 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.

[0160] Cat feces sample preparation . The source of fecal samples was provided by feline animals known to be free of lungworm infection or infected with lungworms. A sample (approximately 1 gram) from a frozen, unpreserved cat fecal sample was suspended in 4 ml of diluent solution ("diluent solution" was 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 at 4000 rpm in a tabletop centrifuge for 20 minutes to produce a first supernatant. The first supernatant was centrifuged at 10,000 g for 5 minutes to produce a second supernatant, which is referred to herein as the "fecal extract".

[0161] Cat serum sample preparation. The source of serum samples was provided by cat animals known to be free of lungworm infection or infected with lungworm. Serum samples from six experimentally infected cats were obtained from the laboratory of Dr. Bowman of Cornell University and used throughout the study. Cats were infected at about 12-14 weeks of age. Each cat received 250 third-stage larvae (L3) harvested from the smooth double-umbilical snail (Biomphalaria glabrata) aquatic snail colony in the laboratory to start their infection. Fecal samples were collected weekly, and the Baermann procedure was performed to calculate the larval shedding rate (in terms of larvae per gram) of each cat. Blood was also drawn weekly, wherein samples were taken from alternating medial saphenous veins (alternating medial saphenousveins). Experimental infection lasted until the 89th day, at which time point imidacloprid (Imidacloprid) and Moxidectin (Moxidectin) therapy (Advantage Multi, Bayer) was introduced. In a two-month course of treatment, cats received at least two doses of the therapy. Stool collections and blood draws continued weekly until day 189.

[0162] Recombinant TDX1557 protein preparation A codon-optimized version of the 2M2 gene was synthesized ( SEQ ID NO:19 ) and cloned into pCDNA 3.1 (TDX1557).

[0163] 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 part B of FIG. 1 ) for purification of recombinant TDX1557.

[0164] When preparing the construct (SEQ ID NO: 20), the IgG-like signal sequence was encoded by MDWTWRVFFLLALATGVHSEN The sequence of (SEQ ID NO:21) is included in the amino terminal region of 2M2 (positions 941-1003 of SEQ ID NO:2) to secrete the recombinant 2M2 (rTDX1557) polypeptide (SEQ ID NO:3) into the supernatant. The C-terminal region carries a tag ( AAA HHHHHHHGVLAPHDSVLQ) (SEQ ID NO: 22) to facilitate the purification of the recombinant protein. TMThe recombinant construct was transfected into a mammalian expression system (HEK293 cells) using a transfection kit (Thermo Fisher Scientific, Carlsbad, CA). The supernatant was collected after 5-7 days and the recombinant protein TDX1557 (SEQ ID NO: 3) was purified using immobilized metal affinity chromatography (IMAC).

[0165] Preparation of polyclonal feline lungworm antibodies Polyclonal antibodies "anti-TDX1557pAB" (IgG) were raised in rabbits against the polypeptide having the amino acid sequence corresponding to SEQ ID NO: 3, respectively, using standard methods and purified from serum. After rTDX1557 was introduced into rabbits, anti-TDX1557 pAB was purified from the plasma of immunized rabbits by separating IgG antibodies using protein G affinity chromatography.

[0166] Infections in cats . Parasitic lungworm infection was achieved by oral administration of approximately 150-300 infective Strongyloides felis larvae to healthy cats. Infection was confirmed by microscopic observation of worm eggs in fecal samples obtained from these host animals. Larval shedding was performed using the standard Baermann method (AJCarruth et al.Journal of FelineMedicine and Surgery, 2019, Vol.5, pp.1-6). Infected cats were treated with the antiparasitic drugs imidacloprid and moxidectin on day 90.

[0167] ELISA assay. Lungworm lysate or recombinant TDX1557 ("rTDX1557") (100 μl / well; 3 μg / ml for rTDX1557 and ~2 μg / mL for lysate) were immobilized on Immulon IV 96-well plates overnight at 4°C by physical adsorption. The plates were then blocked with 1% BSA in 0.1 M Tris pH 7.0 for 2 hours at room temperature. The wells were then washed five times with PBS-Tween-20 solution according to standard methods known to those of ordinary skill in the art. Serum samples from experimentally infected cats were diluted with PRRS diluent (IDEXX PRRS X3 Ab Test, Cat. No. 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) at a 1 in 200 dilution (100 μl / well; 1 in 200 dilution) and added to the wells. After incubation at room temperature for one hour, the wells were washed five times with PBS-Tween-20 solution according to standard methods known to those of ordinary skill in the art. Then 50 μl of goat anti-cat IgG (H+L)-HRP antibody (Jackson ImmunoResearch Inc., West Grove, PA) diluted 1 to 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 by standard spectrophotometric techniques to generate an "OD450 value" (or more simply "OD value") for each well. In this arrangement, the OD value obtained for any particular well of the 96-well plate is directly proportional to the amount of specifically bound antibody present in the well.

[0168] Immunoblotting procedures: Worm lysate (3 μg / ml) or recombinant TDX1557 (10 μg) was mixed with SDS-PAGE sample buffer and subjected to SDS-PAGE gel electrophoresis at 200 V for 35 minutes (Thermo Fisher, Carlsbad, MA). Total protein from the lysate of SDS-PAGE gel was transferred to nitrocellulose (NC) membrane using iblot (Thermo Fisher, Carlsbad, CA). The NC membrane was probed with rabbit polyclonal antibody against TDX1557, and then anti-rabbit IgG H+L coupled to alkaline phosphatase (Sigma, St Louis, MO) was added. The blot was developed with BCIP / NBT substrate (Sigma, St Louis, MO).

[0169] ELISA Procedure for Peptide Array Analysis Using Neutravidin Plates . Neutravidin plates (Thermo Fisher, Carlsbad, CA) were coated with 100 μl of peptide diluted in PBS in a Neutravidin plate, and the plates were then incubated with shaking at RT for 1 hour. Afterwards, the plates were washed 3 times with PBS-T. Cat serum samples were diluted 1:800 in 1×PBS (pH 7.2+2% BSA), 100 μl (what is the concentration?) was applied to each well, and the plates were incubated with shaking at RT for 1 hour. Afterwards, the plates were washed 3 times with PBS-T. 100 μl of secondary antibody dilution was added to each well, followed by 12 μl of goat anti-cat H+L HRP in 60 ml ECD. The plates were then incubated with shaking at RT for 1 hour, washed 5 times with PBS-T, and then developed with 100 μl of TMB substrate. Stop solution was added, and the solution was read at 450 nm. Example 1: Preparation and characterization of recombinant TDX1557

[0170] In this example, recombinant TDX1557 (also referred to as recombinant 2M2 or rTDX1557) (SEQ ID NO: 3) was expressed and purified in a transfected HEK293 mammalian cell line as described above. The purified protein was analyzed by SDS-PAGE electrophoresis. The predicted molecular weight of 2M2 (TDX1557) is 28 kDa, but due to post-translational modifications in mammalian expression systems, the molecular weight of recombinant TDX1557 is 62 KDa. To further confirm that recombinant TDX protein was produced, immunoblotting was performed according to the above procedure. The gel was blotted onto a nitrocellulose membrane and probed with an alkaline phosphatase-labeled monoclonal antibody (Sigma, St Louis, MO) against the HIS tag. Substrate NBT / BCIP (Sigma, St Louis, MO) was added according to the above procedure for blot development. The gel and immunoblotting results are shown in Figure 1.

[0171] As shown in Figure 1, part A, SDS-PAGE analysis of supernatants from the mammalian expression system confirmed the expression of recombinant 2M2. Figure 1, part B shows immunoblot analysis of recombinant 2M2. Recombinant 2M2 was probed with mAb HIS labeled with alkaline phosphatase and then developed with BCIP / NBT as described above. Example 2: Detection of lungworm infection using ELISA assay using TDX1557

[0172] This example compares the detection and measurement of lungworm infection using the fecal Baermann technique and an indirect ELISA using lungworm lysate and rTDX1557.

[0173] Experimental infections and procedures in cats :As described above, cats were orally infected with cat strongyloides larvae. Fecal samples and blood samples were collected over a specified time period (0th, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, 56th, 63rd, 70th, 77th, 84th, 91st, 98th, 112th, 126th, 140th, 154th, 168th, and 182nd days). By the 90th day, infected cats were treated with antiparasitic drugs imidacloprid and moxidectin. Standard procedures were used to perform the Baermann method on fecal samples for visual inspection and lungworm larvae counting (AJ Carruthet al., JFMS, 2019, Vol.5, pp.1-6). Serum samples collected over a specified time period were evaluated in an indirect ELISA assay using lungworm lysates and rTDX1557. As described above, cat strongyloides lysates were prepared from lungworm larvae. Total protein in the lysate was determined by conventional Bradford assay. Recombinant protein TDX1557 was obtained as described above.

[0174] ELISA assay of Strongyloides felis lysate and rTDX1557 .

[0175] Fecal samples from experimentally infected samples were used to determine lungworm larval shedding rates by the standard Baermann method. Lungworm lysates and rTDX1557 were subjected to indirect ELISA assays using the above-described procedure. Two cats (BigMac and Burrito) were experimentally infected with lungworms, and serum samples from these cats were diluted with PRRS buffer (IDEXX PRRS X3 Ab Test, Cat. No. 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) and used in the ELISA procedure.

[0176] Lungworm lysate or rTDX1557 was coated overnight at 4°C on Immulon IV plates and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5×PBST, serum samples from experimentally infected cats (BigMAC or Burrito) were diluted with PRRS diluent and added to the plates and kept at RT for one hour. The plates were washed again with 5×PBST, and the secondary antibody (anti-cat IgG H+L) was added to the plates and kept for 30 minutes. The plates were washed, substrate was added and read at 450 nm.

[0177] The results of larval shedding in experimentally infected cats are shown in Figure 2 In parts A and B of the study, larval shedding rates were measured over a period of 182 days. ELISA assays were performed using Strongyloides felis lysate and rTDX1557 (2M2) to compare larval shedding rates in high shedding cats ("BigMac") (see Figure 2 Part A) and larval shedding in low-shedding cats ("Burrito") (see Figure 2 Part B). The X-axis represents the days post infection (DPI). The Y1 and Y2 axes represent the OD values ​​and reflect the shedding rate. The results showed that larval shedding measured by the fecal Baermann method produced inconsistent results, but the ELISA assay using lungworm lysate and rTDX1557 showed a gradual increase in antibody titers against lungworms, but the antibody titers decreased after day 90, indicating that the treated cats responded to the treatment. Compared with the ELISA assay using lungworm lysate, the ELISA assay using recombinant TDX1557 showed a much more robust antibody response. Example 3: Anti-TDX1557 has no cross-reactivity with heartworms

[0178] This example demonstrates that rabbit polyclonal antibodies raised against rTDX1557 do not cross-react with heartworm (Dirofilaria immitis) using SDS-PAGE and immunoblot analysis.

[0179] SDS-PAGE and immunoblot analysis of Strongyloides felis and Heartworm lysates .

[0180] Rabbit polyclonal antibodies, heartworm lysate and lungworm lysate for rTDX1557 were prepared as described above. rTDX1557, lungworm lysate and heartworm lysate were each mixed with SDS-PAGE buffer and SDS-PAGE gel electrophoresis was performed on two different gels using conventional procedures. In the first gel, rTDX1557 (lane 1), cat strongyloid lungworm lysate (lane 3) and heartworm lysate (lane 4) were stained with Imperial. The second gel with the same lane was immunoblotted by transferring the total protein of the lysate to a nitrocellulose (NC) membrane. The NC membrane was probed with a rabbit polyclonal antibody for TDX1557, and then an alkaline phosphatase conjugate (anti-rabbit IgG H+L coupled to alkaline phosphatase) (Sigma, St Louis, MO) was added. The blot was developed with BCIP / NBT substrate. The positive control was rTDX1557 with a molecular weight of about 62kDA. Lane 2 was left blank. The results are shown in Figure 3 of parts A and B.

[0181] like Figure 3 As shown in part A, SDA-PAGE analysis of the cat lung nematode Strongyloides felis and Heartworm lysates showed the expression of 2M2 (TDX1557) in cat lung nematode. Figure 3 Part B of FIG. 4 shows an immunoblot analysis of cat strongyloides lysate and heartworm lysate after probing with a rabbit polyclonal antibody against TDX1557 (anti-TDX1557). The rabbit polyclonal antibody against TDX1557 recognized a single band of ∼62 kDa in cat strongyloides lysate but did not cross-react with heartworm lysate. Example 4: Anti-TDX1557 has no cross-reactivity with Ascaris

[0182] This example demonstrates that rabbit polyclonal antibodies raised against rTDX1557 do not cross-react with roundworms (Toxocara Spp) using SDS-PAGE and immunoblot analysis.

[0183] SDS-PAGE and immunoblot analysis of Strongyloides felis and Ascaris lysates.

[0184] Rabbit polyclonal antibodies, roundworm antigens (Toxocara Spp) and lungworm antigens against rTDX1557 were prepared as described above. In an SDS-PAGE gel, rTDX1557 (lane B), cat roundworm lungworm lysate (lane C) and roundworm lysate (lane D) were each mixed with SDS-PAGE buffer and subjected to SDS-PAGE gel electrophoresis using conventional procedures. The resulting gel was subjected to immunoblot analysis by transferring the total protein in the gel to a NC membrane. The NC membrane was probed with a rabbit polyclonal antibody against TDX1557, and then an alkaline phosphatase conjugate (anti-rabbit IgG H+L coupled to alkaline phosphatase, Sigma, St.Louis, MO) was added. The blot was developed with BCIP / NBT substrate (Sigma, St.Louis, MO). The positive control was rTDX1557. The results are shown in Figure 4 middle.

[0185] like Figure 4 , shows immunoblot analysis of Strongyloides cati and Ascaris lysate probed with rabbit polyclonal antibody against TDX1557 (anti-TDX1557) prepared as described above. The rabbit polyclonal antibody against TDX1557 recognized a single band of ∼62 kDa in the Strongyloides cati lysate pellet (lane C), but did not cross-react with Ascaris soluble antigen (lane D). Example 5: ELISA and Tests to detect lungworm infection

[0186] This example compares the detection and measurement of lungworm infection using the fecal Baermann technique (results taken from Example 2) and the indirect ELISA assay (results taken from Example 2) with the SNAP diagnostic device using lungworm lysate and rTDX1557 as a positive control.

[0187] Experimental infection of cats: Cats (BigMac) were orally infected with Strongyloides felis larvae as described in Example 2. Fecal and blood samples were collected over a specified 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). By day 90, infected cats were treated with antiparasitic drugs imidacloprid and moxidectin. Fecal samples were subjected to the Baermann method for visual inspection and lungworm larvae counting using standard procedures (AJ Carruth et al., Journal of Feline Medicine and Surgery, 2019, Vol. 5, pp. 1-6). Serum samples collected over a specified time period were evaluated in an indirect ELISA assay using lungworm lysate and rTDX1557 as a control and in a SNAP assay device using rTDX1557 as a control. Strongyloides felis 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. ELISA assay of Strongyloides felis lysate and rTDX1557 .

[0188] Indirect ELISA assays were performed on lungworm lysates and rTDX 1557 using the procedure described above.Two cats (BigMac and Burrito) were experimentally infected with lungworms and serum samples from these cats were diluted with PRRS buffer and used in the ELISA procedure.

[0189] Lungworm lysate or rTDX1557 was coated on Immulon IV plates overnight at 4°C and the plates were blocked with blocking solution (1% BSA in 100mM Tris pH 7.5). The plates were washed with 5×PBST, serum samples from experimentally infected cats (BigMAC) were diluted with PRRS diluent and added to the plates and kept at RT for one hour. The plates were washed again with 5×PBST, and the secondary antibody (anti-cat IgG H+L) was added to the plates and kept for 30 minutes. The plates were washed, substrate was added and read at 450nm. The results of the indirect ELISA assay using lungworm lysate and rTDX1557 as controls are shown in Figure 5 A (copied from Figure 2 A). SNAP assay

[0190] Recombinant TDX1557 was passively coated (1% solids) at 1 mg / mL in PBS onto polystyrene particles [Sphero, Lake Forest, IL] and 0.7 μl was spotted onto the matrix [IDEXX Laboratories]. The positive control was goat anti-HRPO coated latex particles. The SNAP device was built with wash buffer (IDEXX Laboratories). Recombinant TDX1557 was also covalently linked to HRPO via periodate conjugation. The conjugate (180 μl) was mixed with 150 μl of serum sample and added to the sample well. Once the sample reached the activation circle, the SNAP device was activated to wash the matrix and then developed with substrate. SNAP results were read visually and by densitometer. SNAP results are shown in Figure 5 The results of larval shedding and indirect ELISA assays are summarized in Figure 5 Part A (copied from Figure 2 Part A). Figure 5 As shown in Part A of , comparison of fecal results using the standard Baermann test showed inconsistent larval shedding relative to the indirect ELISA results using serum samples, in which antibody responses to rTDX1557 and Strongyloides felis lysate showed steady progress over this time period.

[0191] The SNAP results using serum samples are summarized in Figure 5 In section C. Antibody reactivity was observed from day 14 onwards.

[0192] Numerical comparison of the Baermann test results (larval shedding), indirect ELISA (using rTDX1557), and SNAP results is shown in Figure 5 In section B of . Relative to larval shedding and indirect ELISA assays, SNAP assay results should detect lungworm infection as early as day 14 post-experimental infection. In contrast, indirect ELISA and larval testing showed detection of lungworm infection on days 35 and 63 post-experimental infection. SNAP results for ten cat field samples showed good correlation with indirect ELISA negatives, with only one exception. Example 6: Construction, expression and characterization of truncated versions of TDX1557

[0193] This example discusses the construction of truncated versions of 2M2 / TDX1557 and the evaluation of these truncated 2M2 peptides in an indirect ELISA assay using rabbit anti-2M2 pAbs, temporal (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 As discussed above for rTDX1557, truncated versions of TDX1557 labeled T2 (also known as 2M2_T2 or TDX1637), T3 (also known as 2M2_T3 or TDX1674), and T4 (also known as 2M2_T4 or TDX1675) were cloned and expressed. Construct for T2 (SEQ ID NO: 23)

[0194] Construct for T3 (SEQ ID NO: 24)

[0195] Construct for T4 (SEQ ID NO: 25)

[0196]

[0197] When preparing the constructs, an IgG-like signal sequence was inserted into the amino-terminal region of each truncation. MDWTWRVFFLLALATGVHSEN (SEQ ID NO:21) is included at positions 1 to 63 of SEQ ID NO:23 (T2 construct), positions 1 to 63 of SEQ ID NO:24 (T3 construct), and positions 1 to 63 of SEQ ID NO:25 (T4 construct) to secrete the truncation into the supernatant. The C-terminal region is tagged with a HIS tag ( AAAHHHHHHHGVLAPHDSVLQ) (SEQ ID NO: 22) tag was used to facilitate the purification of the recombinant truncated protein. The recombinant construct was transfected into a mammalian expression system (HEK293 cells) using the ExpiFectamineTM transfection kit (Thermo Fisher Scientific, Carlsbad, CA). The supernatant was collected after 5-7 days and the recombinant truncated protein was purified using immobilized metal affinity chromatography (IMAC). The predicted structure [https: / / wlab.ethz.ch / protter / ] indicated the secondary structure of the truncated version, such as helices and β-folds. SDS-PAGE analysis showed the expression of each truncated TDX1557 protein.

[0198] 2M2(TDX1557)

[0199] 2M2_T2

[0200] >2M2_T3

[0201] >2M2_T4

[0202] The indirect ELISA procedure used in this example is as described above. Briefly, rTDX1557 and recombinant truncations T2, T3, and T4 were each coated overnight at 4°C on separate Immulon IV plates and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5×PBST, serum samples from experimentally infected cats were diluted with PRRS diluent and added to the plates, and the plates were kept at RT for 1 hour. The plates were washed again with 5×PBST, and the secondary antibody (anti-cat IgG H+L) was added to the plates and kept for 30 minutes. The plates were washed, substrate was added and read at 450 nm. The results are shown in Figure 8 middle.

[0203] Figure 8Evaluation of truncated versions of 2M2 (TDX1557) using an indirect ELISA assay is shown. Four (4) different versions of the recombinant peptide (2M2 full, T2, T3, and T4) were evaluated in an indirect ELISA using rabbit pAb, temporal (Burrito DPI 98, French Fry DPI 91), and negative samples (SPF 16CMF3, SPF16CSL3). Serially diluted serum samples were tested at different concentrations (0.5, 1.5, and 3 μg / ml) of the recombinant peptide. Rabbit polyclonal antibodies against TDX1557 reacted with all four different recombinant peptides. However, experimentally infected samples did not show any reactivity with the 2M2 T3 truncate, indicating that the important epitope is only present at the carboxyl terminus of the protein. Specific pathogen-free (SPF) negative controls (SPF Cat ID 16CMF3 and 16CSL3) showed no reactivity to any of the recombinant peptides. An increase in titer was also observed based on antigen concentration and dilution rate, indicating an antibody response against the truncated version.

[0204] The truncations 2M2 Amino2 Cys, 2M2 CpepC, 2M2 Wo epitope, and truncated Cys-Ser variants of 2M2 T4 (designated 2M2 T4 WO IDR, 2M2 T4 1A, 2M2 T4 1B, and 2M2 T4 1C) were also constructed. Fig.16C1 The sequences of these truncations are shown in Fig.16C2 The truncations 2M2 Amino2 Cys and 2M2 CpepC were successfully expressed, but the truncated Cys-Ser variant was not expressed. Fig.16D and 16E The evaluation of 2M2 Amino2Cys, 2M2 CpepC, and 2M2 Woepitope using the above procedure is shown. Fig.16D and 16E As shown, no reactivity was found to the temporal samples. Example 7A: Identification of epitopes of TDX1557 using peptide arrays

[0205] This example shows the evaluation of a peptide array using serum samples from uninfected cats (TDX 1557 ELISA negative) based on the ELISA protocol on neutravidin plates as described above. The array contained 62 15mer peptides covering the entire full-length 2M2 protein with 10 amino acid overlaps.

[0206]

[0207] These peptides were synthesized using standard procedures (New England peptide, MA) and labeled with biotin at the N-terminus. The biotinylated peptides were coated on neutravidin plates (Thermofisher, Carlsbad, CA). The plates were incubated with serum samples and then washed with PBST. A secondary antibody (anti-cat Ig G H+L) conjugated with HRP was added and kept 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 450nm.

[0208] like Fig. 9 As shown, negative samples (AC03772, AC04400, and AN06876) and SPF Pool samples were used. The raw data were tabulated directly between the negative and positive samples to show the differences. For experimental or temporal samples, the OD value on day 0 (before blood sampling) was used to normalize the data for that specific cat. Fig.10 Positive field samples in (AC03791, AC03796, AC12570, AC12692, AC12693, AN00997, AN24140, and BB62456) or Fig.11 Compared to the experimentally infected samples in (BigMac 91dpi, Burrito 85dpi, Nugget 85dpi, Taco dpi85 and Tortilladpi85), the negative samples showed no significant reactivity to the peptides. Example 7B: Identification of epitopes of TDX1557 using peptide arrays

[0209] This example shows the evaluation of the peptide array of Example 7A using serum samples from naturally infected cats (TDX 1557 ELISA positive) using the ELISA protocol on neutravidin plates as described above. The protein array was evaluated using serum samples from naturally infected cats that were positive in the ELISA assay using intact protein. The results showed significant reactivity to the peptides.

[0210] The N-terminal biotinylated peptide of Example 7A was coated in a Neutravidin plate using the above procedure. The plate was incubated with serum samples and then washed with PBST. A secondary antibody (anti-cat Ig G H+L) with HRP conjugate was added and kept at room temperature for 30 minutes. The plate was washed with PBST and the substrate was added. Stop solution was added and the solution was read at 450nm. The results are shown in Fig.10 middle.

[0211] like Fig.10As shown, positive cat serum samples showed significant reactivity to the following peptide sequences. These peptide sequences were found to be the most immunogenic peptides for TDX1557:

[0212] Example 7C: Identification of immunodominant epitopes of TDX1557 using peptide arrays

[0213] This example shows the evaluation of the peptide array of Example 7A using serum samples from experimentally infected cats (TDX 1557 ELISA positive) using the ELISA protocol on neutravidin plates as described above.

[0214] The N-terminally biotinylated peptides of Example 7A were coated on Neutravidin plates (Thermofisher, Carlsbad, CA) using the procedure described above. The plates were incubated with serum samples and then washed with PBST. A secondary antibody (anti-cat IgG H+L) with an HRP conjugate was added and kept 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 middle.

[0215] like Fig.11 As shown, the peptide array of 2M2 was evaluated with eight positive experimental infection samples (Big Mac 91dpi, Burrito 85dpi, Nugget 85dpi, Taco 85dpi, Tortilla 85dpi) and a specific pathogen-free (SPF) cat pool (negative control). Experimental infection samples that showed significant positive reactivity in the ELISA assay using intact protein 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 have the highest immunogenicity.

[0216] Fig.11 Also included is an alignment of D6, D7, and D8, where the underlined overlap region shared by these three peptides, with the sequence SKSWF (SEQ ID NO:81) comprising the epitope. The overlap region between D6 and D7 indicates that the sequence MATRASKSWF (SEQ ID NO:82) comprises the epitope. The overlap region between D7 and D8 indicates that the sequence SKSWFDELKK (SEQ ID NO:83) comprises the epitope.

[0217] Fig.11Also included is an alignment of C10, C11, and C12, where the underlined overlap region shared by these three peptides indicates that the sequence AMIHA (SEQ ID NO:84) contains the epitope. The overlap region between C10 and C11 indicates that the sequence NVEKTAMIHA (SEQ ID NO:85) contains the epitope. The overlap region between C11 and C12 indicates that the sequence AMIHAKKCVF (SEQ ID NO:86) contains the epitope.

[0218] Fig.11 Also included is an alignment of D2, D3, and D4, where the overlapping region shared by the three peptides with the sequence ENVWV (SEQ ID NO: 87) contains the epitope. The overlapping region between D2 and D3 indicates that the sequence RKDTGENVWV (SEQ ID NO: 88) contains the epitope. The overlapping region between D3 and D4 indicates that the sequence ENVWVMWPAQ (SEQ ID NO: 89) contains the epitope. Example 8: Evaluation of polypeptides D6, D7 and D8

[0219] This example shows a comparison of the reactivity of D6, D7 and D8 peptides, Strongyloides felis lysate and rTDX1557 with antisera from experimentally infected cats. The peptides (D6, D7, D8) were prepared as described in Example 7A. Antibody binding of D6, D7 and D8 peptides, cat roundworm lysate and rTDX1557 was measured using an indirect ELISA assay. Linear peptides (D6, D7, D8) bound antibodies at early time points, indicating that they have diagnostic utility for detection and diagnosis of infection, including early stages of infection. ELISA assays with larval lysate and rTDX1557 showed a gradual increase over time, consistent with the gradual increase in antibody titers. Antibody titers decreased after antiparasitic drug treatment, which occurred at DPI 90. The OD values ​​are shown in the table below.

[0220]

[0221] Fig.12 Shown is a comparison of ELISA assays of peptides (D6, D7, and D8), recombinant TDX1557, and Strongyloides felis lungworm lysate using serum samples from experimentally infected high-shedding cats Tortilla. Example 9: Evaluation of D678 polypeptide in infected cats

[0222] This example describes the construction of the D678 polypeptide and its evaluation of its reactivity with serum samples taken from experimentally infected cats.

[0223] Fig.13 Shows the use of Protter( https: / / wlab.ethz.ch / protter / ) (an open source tool for visualizing protein forms) to construct the polypeptide structure of 2M2. Protter indicated that 2M2 is a secreted protein and the underlined region containing the immunogenic peptides D6, D7 and D8 within the 2M2 sequence. Alignment of D06, D07 and D08 showed overlapping SKSWF sequences (SEQ ID NO:81). Combination peptide D678 ("D678 combination") was synthesized based on the combination of D6, D7 and D8 peptides using standard peptide synthesis procedures.

[0224] CPAQKNLTEMATRASKSWFDELKKFG D678 combination (SEQ ID NO: 18)

[0225] The N-terminal C residue of SEQ ID NO: 18 is not part of the native sequence. It is added for ease of manipulation (e.g., conjugation), as is well known in the art. "Modified D678", hereinafter SEQ ID NO: 78, is identical to SEQ ID NO: 18 except that it does not have the N-terminal C.

[0226] The cat nematode lung nematode lysate prepared as described above, rTDX1557 prepared as described above, and non-biotinylated synthetic peptide D678 were separately coated on Immulon IV plates overnight at 4°C, and the plates were blocked with blocking solution (1% BSA in 100mM Tris pH7.5). The plates were washed with 5×PBST, and serum samples from experimentally infected cats were diluted with PRRS diluent and added to the plates and incubated for one hour at RT. The plates were washed again with 5×PBST, and the secondary antibody (anti-cat IgG H+L) was added to the plates and incubated for 30 minutes at RT. The plates were washed, the substrate was added, and the plates were read at 450nm. The results of the indirect ELISA assay using lung nematode lysate and rTDX1557 as controls and peptide D678 are shown in Fig.14 middle.

[0227] Although peptide D678 is a combined version of the immunogenic D6, D7 and D8 peptides (which generated early antibody responses, as discussed and shown in Examples 8 and 9), it was found that peptide D678 was not equivalent in its antibody response relative to the individual peptides (D6, D7 and D8). The lack of high antibody response may be due to the fact that a non-biotinylated form of D678 was evaluated relative to the biotinylated D6, D7 and D8 peptides used in the experiments discussed above. Example 10: Construction of extended D678 polypeptides and evaluation for detection of antibodies from infected cats

[0228] In this example, two extended versions of the D678 peptide were constructed, in which the immunogenic region was extended at the amino terminus (D678 amino) or the carboxyl terminus (D678 carboxyl) and evaluated by indirect ELISA assay to determine whether either or both of the extended D678 peptides would exhibit enhanced immunoreactivity relative to the modified D678 peptide. An alignment of the amino acid sequences of the modified D678 [SEQ ID NO: 78], D678 amino [SEQ ID NO: 79], and D678 carboxyl [SEQ ID NO: 80] peptides is shown in Fig.15 The overlapping amino acids of the modified D678 peptide and the extended versions of the peptide (D678 amino and D678 carboxyl peptides) are underlined. D678, D678 amino peptide, and D678 carboxyl peptide were synthesized by New England Peptide (Gardner, MA).

[0229] Modified D678: PAQKNLTEMATRASKSWFDELKKFG (SEQ ID NO:78)

[0230] D678 amino:

[0231] D678 Carboxyl: The N-terminal C residue in SEQ ID NO: 80 was added to facilitate conjugation, ie it is not part of the native sequence.

[0232] The indirect ELISA procedure used in this embodiment is as described above. In short, the recombinant modified D678, D678 amino, D678 carboxyl peptides were coated overnight on Immulon IV plates at 4 ° C, and then the plates were blocked with blocking solution (1% BSA in 100mM Tris pH 7.5). The plates were washed with 3 × PBST, and 100 μl serum samples (1:100 diluted samples) from infected cats were diluted with PRRS diluent (IDEXX PRRS X3 Ab Test, Cat. No. 99-18070, IDEXX Laboratories, Inc., Westbrook, ME, USA) and added to the plates, and the plates were kept at RT for one hour. The plates were washed with 1 × PBS-Tween buffer, aspirated and conjugates were added to 100 μl / well (diluted 1:3000 with enzyme conjugate diluent). Conjugates (anti-cat IgG H+L) were added to the plates and incubated at RT for 30 minutes. The plate was washed, 50 μl / well TMB substrate was added and incubated for 5 minutes at RT. 50 μl / well stop solution was added. The wells were read at 450 nm. The results are shown in FIG16 .

[0233] Fig.16A Results of an indirect ELISA format for evaluating the extended D678 peptide using temporal, rabbit pAb, and SPF cats are provided. Synthetic peptides D678 amino and D678 carboxyl showed similar reactivity to samples from temporal samples, indicating that the extended region does not increase the immunogenicity of the peptide. Although the rabbit pAb for TDX1557 did not show any antibody response to the D678 peptide, the rabbit pAb reacted to both the extended D678 amino and D678 carboxyl peptides. D678, D678 amino and D678 carboxyl peptides did not show any significant reactivity to serum samples from experimentally infected cats. Example 11: Detection of lungworm antigen (2M2 antigen) in fecal samples using rabbit polyclonal antibodies against TDX1557

[0234] In this example, fecal samples collected from an experimentally infected cat (Big Mac) at DPI 36, DPI 64, DPI 141, and DPI 180 were compared with Giardia diluent (IDEXX Laboratories, Westbrook, ME) was mixed at a ratio of 10% (0.5 g sample in 5 ml diluent). The suspension was mixed and centrifuged at 10,000g for 5 minutes. 100 μl of 2×SDS-PAGE sample buffer was added to 100 μl of supernatant, and the mixture was kept in a heating block (90°C) for 10 minutes. The mixture was subjected to SDS-PAGE electrophoresis using conventional procedures. The resulting gel was immunoblotted by transferring the total protein from SDS-PAGE to a nitrocellulose (NC) membrane. The NC membrane was probed with a rabbit polyclonal antibody against TDX1557, and then an alkaline phosphatase conjugate (anti-rabbit IgG H+L coupled to alkaline phosphatase, Sigma, St. Louis, MO) was added. The blot was developed with BCIP / NBT substrate (Sigma, St. Louis, MO). The positive control was rTDX1557. The negative control was a fecal sample from a healthy, uninfected cat. Fig.17Detection of Strongyloides felis antigen (2M2) in fecal samples of experimentally infected cats BigMac is shown. The results demonstrate that a 62 kDA band was detected in fecal samples of BigMac at DPI 36, DPI 64, and DPI 141 using a rabbit polyclonal antibody against TDX1557. The band was not detected at DPI 180. Despite treatment of experimentally infected cats with antiparasitic drugs at DPI 90, 2M2 antigen was still detectable in fecal samples until DPI 140. However, the 2M2 antigen completely disappeared by DPI 180. The positive control was recombinant 2M2 antigen (rTDX1557) and the negative control was a fecal sample from a healthy, uninfected cat. Example 12: Alanine scanning of the 2M2 epitope

[0235] This example shows the effect of amino acid substitutions in the regions defined by peptides D6, D7 and D8 on antibody binding. Alanine scanning was performed for this purpose. As shown in the table below, alanine (bold letters) was used to replace the native amino acids in peptides D7M (SEQ ID NO: 90) and D8M (SEQ ID NO: 106). Where alanine already existed in the native sequence, it was replaced with glycine.

[0236] Table: D7M peptide

[0237] Table: D8M peptide SEQ ID NO: Peptide ID Peptide sequence 106 D8M0 TRASKSWFDELKKFG 107 D8M1 ARASKSWFDELKKFG 108 D8M2 TAASKSWFDELKKFG 109 D8M3 TRGSKSWFDELKKFG 110 D8M4 TRAAKSWFDELKKFG 111 D8M5 TRASASWFDELKKFG 112 D8M6 TRASKAWFDELKKFG 113 D8M7 TRASKSAFDELKKFG 114 D8M8 TRASKSWADELKKFG 115 D8M9 TRASKSWFAELKKFG 116 D8M10 TRASKSWFDALKKFG

[0238] Peptides were synthesized using standard procedures and labeled with biotin at the N-terminus.

[0239] If a large decrease in OD values ​​is observed, indicating that the replaced amino acid is essential for proper antibody binding, amino acids that are critical for antibody binding can be detected. In addition, if there is an increase in OD values ​​compared to the original peptide, the experiment provides an opportunity to identify favorable substitutions.

[0240] The indirect ELISA assay was performed as described in Example 7A to test the immunoreactivity of peptides D7M0-D7M15 and D8M0-D8M10 with pooled sera from three cats naturally infected with Strongyloides felis (i.e., field positive). The assay was performed in triplicate. The OD values ​​were normalized to the immunoreactivity (OD values) with sera from day 0 samples of experimentally infected cats.

[0241] The results are shown in Fig.18Compared with D8M0, the OD values ​​of D8M7 (tryptophan changed to alanine) and D8M8 (phenylalanine changed to alanine) were strongly reduced, indicating that amino acid positions 7 and 8 are critical for the binding of antibodies in sera from cats infected with cat Strongyloides. Compared with D8M0, the OD value of D8M10 increased, indicating that replacing glutamic acid (a negatively charged amino acid) with alanine (a small non-polar amino acid) enhanced the binding of the peptide to antibodies in sera from cats infected with cat Strongyloides. Similar results were obtained when the peptides were tested with sera from cats experimentally infected with cat Strongyloides at different time points (data not shown).

[0242] Thus, the amino acids corresponding to position 7 (tryptophan) and position 8 (phenylalanine) of peptide D8M0 (SEQ ID NO: 106) are critical for binding of antibodies from cats infected with Strongyloides felis. These amino acid positions correspond to position 4 (tryptophan) and position 5 (phenylalanine) of a peptide having the sequence SKSWF (SEQ ID NO: 81).

[0243] The data indicate that the consensus sequence of the core epitope is XXXWF (SEQ ID NO: 117). Therefore, in one embodiment of the present disclosure, the polypeptide comprises XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2 and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2 and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids.

[0244] Some embodiments of the present disclosure include:

[0245] Embodiment 1: A polypeptide comprising the amino acid sequence 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (D678-amino extension), 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 XXXWF (SEQ ID NO:117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0246] Embodiment 2: The polypeptide according to embodiment 1, wherein the polypeptide is selected from the group consisting of the following 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D678), SEQ ID NO: 80 (modified D678), SEQ ID NO: 81 (modified D678), SEQ ID NO: 82 (modified D678), SEQ ID NO: 83 (modified D678), SEQ ID NO: 84 (modified D678), SEQ ID NO: 85 (modified D678), SEQ ID NO: 86 (modified D678), SEQ ID NO: 87 (modified D678), SEQ ID NO: 88 (modified D678), SEQ ID NO: 89 (modified D678), SEQ ID NO: 90 (modified D678), SEQ ID NO: 91 (modified D678), SEQ ID NO: 92 (modified D678), SEQ ID NO: 93 ( NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0247] Embodiment 3: The polypeptide according to embodiment 1, wherein the polypeptide comprises at least 5 consecutive amino acids of the following amino acid sequence: 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D778), SEQ ID NO: 80 (modified D778), SEQ ID NO: 81 (modified D778), SEQ ID NO: 82 (modified D778), SEQ ID NO: 83 (modified D778), SEQ ID NO: 84 (modified D778), SEQ ID NO: 85 (modified D778), SEQ ID NO: 86 (modified D778), SEQ ID NO: 87 (modified D778), SEQ ID NO: 88 (modified D778), SEQ ID NO: 89 (modified D778), SEQ ID NO: 90 (modified D778), SEQ ID NO: 91 (modified D778), SEQ ID NO: 92 (modified D778), SEQ ID NO: 9 NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0248] Embodiment 4: The polypeptide according to embodiment 1, 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 the full length of amino acids of TDX1557. Alternatively, the polypeptide according to embodiment 1, wherein the polypeptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids as shown in SEQ ID NO:2-SEQ ID NO:18 and SEQ ID NO:78-SEQ ID NO:117.

[0249] Embodiment 5: The polypeptide according to embodiment 1, wherein the polypeptide has 10-25 connected amino acids and comprises at least 5, 6, 7, 8, 9 or 10 consecutive amino acids shown in the following 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 combination), 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 (d19 peptide), SEQ ID NO: 33 (d19 peptide), SEQ ID NO: 34 (d11 peptide), NO:78 (modified d678), SEQ ID NO:79 (d678-amino extension), 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 XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2, and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ;

[0250] Embodiment 6: The polypeptide according to embodiment 1, wherein the peptide is labeled or attached to a solid support.

[0251] Embodiment 7: A method for detecting the presence or absence of one or more feline lungworm antibodies in a biological sample from a mammal, the method comprising: (a) contacting the sample with one or more polypeptides according to any one of embodiments 1-6, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; and (c) detecting the presence or absence of the antibody-polypeptide complex, if any.

[0252] Embodiment 8: A method for diagnosing whether a mammal is infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from the mammal with one or more polypeptides according to any one of embodiments 1-6, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; (c) detecting the presence or absence of the antibody-polypeptide complex, if any; and (d) diagnosing the mammal as having Feline lungworm if the antibody-polypeptide complex is present.

[0253] Embodiment 9: A method for diagnosing and treating a mammal infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from a cat with one or more polypeptides according to any one of embodiments 1-6, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; (c) detecting the presence or absence of the antibody-polypeptide complex, if any; (d) diagnosing the mammal as having feline lungworm if the antibody-polypeptide complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat said mammal having a feline lungworm infection.

[0254] Embodiment 10: A method for detecting the presence or absence of one or more feline lungworm antigens in a biological sample, the method comprising: (a) contacting a sample from a cat with one or more antibodies that specifically bind to one or more polypeptides according to any one of embodiments 1-6; (b) forming an antibody-antigen complex in the presence of said one or more feline lungworm antigens, if any, in said sample; and (c) detecting the presence or absence of the antibody-antigen complex, if any.

[0255] Embodiment 11: A method for diagnosing whether a mammal is infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from the mammal with one or more antibodies that specifically bind to one or more polypeptides according to any one of embodiments 1-6; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if any, in the sample; (c) detecting the presence or absence of the antibody-antigen complex, if any; and (d) if the antibody-antigen complex is present, diagnosing the mammal as having feline lungworm.

[0256] Embodiment 12: A method for diagnosing and treating a mammal infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from a mammal with one or more antibodies that specifically bind to one or more polypeptides according to any one of embodiments 1-6; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if any, in the sample; (c) detecting the presence or absence of the antibody-antigen complex, if any; (d) diagnosing the mammal as having feline lungworm if the antibody-antigen complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat said mammal having a feline lungworm infection.

[0257] Embodiment 13: A method according to embodiment 9 or 12, wherein step (e) further comprises one or more additional therapeutic agents to (a) treat an infection caused 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.

[0258] Embodiment 14: A method according to any one of embodiments 8, 9, 11 or 12, wherein step (d) also includes one or more therapeutic agents to (b) control, eliminate or kill the intermediate host of the cat lungworm parasite, flatworm parasite, helminth parasite, non-helminth parasite, virus, fungus, protozoa or bacteria.

[0259] Embodiment 15: The method of any one of embodiments 1-14, wherein the one or more antibodies bind to Aelurostrongylus abstrusus but do not bind to antibodies to Capillaria aerophila or Troglostrongylus brevior.

[0260] Embodiment 16: The method of embodiments 7-9, wherein the one or more polypeptides do not bind to antibodies to roundworms, tapeworms, whipworms or hookworms.

[0261] Embodiment 17: The method of embodiments 10-12, wherein the one or more antibodies do not bind to antigens of roundworms, tapeworms, whipworms, or hookworms.

[0262] Embodiment 18: The method according to embodiment 16 or 17, wherein the ascaris is of the genus Toxocara.

[0263] Embodiment 19: The method according to any one of embodiments 16-18, wherein the tapeworm is Taenia or Diphylidium.

[0264] Embodiment 20: The method of any one of embodiments 16-19, wherein the hookworm is of the genus Ancylostoma.

[0265] Embodiment 21: A method according to any one of embodiments 7-20, wherein step (c) of detecting the presence or absence of the complex further comprises the step of providing at least one second antibody that binds to one or more complexes.

[0266] Embodiment 22: The method according to embodiment 21, wherein the second antibody is labeled or attached to a solid support.

[0267] Embodiment 23: A method according to embodiment 22, wherein the solid support forms part of an enzyme-linked immunosorbent assay device.

[0268] Embodiment 24: The method according to embodiment 23, wherein the enzyme-linked immunosorbent assay device is a lateral flow immunoassay device.

[0269] Embodiment 25: The method of any one of embodiments 1-24, wherein the mammal is a cat.

[0270] Embodiment 26: A polynucleotide encoding a polypeptide according to any one of embodiments 1-6.

[0271] Embodiment 27: A polynucleotide according to embodiment 26, wherein the polynucleotide is RNA.

[0272] Embodiment 28: The polynucleotide of embodiment 27, wherein the polynucleotide comprises one or more modified uridine residues.

[0273] Embodiment 29: The polynucleotide of embodiment 28, wherein the modified uridine residue is pseudouridine or methylpseudouridine.

[0274] Embodiment 30: A polynucleotide according to embodiment 26, wherein the polynucleotide is mRNA.

[0275] Embodiment 31: A polynucleotide according to embodiment 30, wherein the polynucleotide is contained in a lipid nanoparticle.

[0276] Embodiment 32: A vaccine composition comprising one or more polynucleotides according to any one of embodiments 26-31 and a pharmaceutically acceptable carrier.

[0277] Embodiment 33: A vaccine composition comprising one or more polypeptides according to any one of embodiments 1-6 and a pharmaceutically acceptable carrier.

[0278] Embodiment 34: An immune complex comprising one or more polypeptides according to any one of embodiments 1-6 and one or more feline lungworm antibodies that specifically bind to the one or more polypeptides.

[0279] Embodiment 35: An immune complex comprising one or more antibodies that specifically bind to one or more polypeptides according to any one of embodiments 1-6 and one or more feline lungworm antigens that specifically bind to the one or more antibodies.

[0280] Embodiment 36: A device for detecting the presence or absence of one or more feline lungworm antibodies from a biological sample, the device comprising a solid support, wherein the solid support has one or more polypeptides according to any one of embodiments 1-6 immobilized thereon.

[0281] Embodiment 37: The device of embodiment 36, wherein the one or more polypeptides do not specifically cross-react with one or more antibodies to roundworms, tapeworms, hookworms, aerophilic capillaris, or brachysinus strongyloides.

[0282] Embodiment 38: The device according to embodiment 37, wherein the ascaris is of the genus Toxocara; and / or the tapeworm is of the genus Taenia or Dipylidium; and / or the hookworm is of the genus Ancylostoma.

[0283] Embodiment 39: A device for detecting the presence or absence of one or more feline lungworm antigens from a biological sample, the device comprising a solid support, wherein the solid support has one or more antibodies immobilized thereon, and the one or more antibodies specifically bind to one or more polypeptides according to any one of embodiments 1-6.

[0284] 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, aerophilic capillaris, or brachysinus strongyloides.

[0285] Embodiment 41: The device according to embodiment 40, wherein the ascaris is of the genus Toxocara; and / or the tapeworm is of the genus Taenia or the genus Dipylidium; and / or the hookworm is of the genus Uncinaria.

[0286] Embodiment 42: A method of immunizing a mammal (eg, a cat) against feline lungworm, the method comprising administering the vaccine composition of 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 from a mammal, the method comprising: (a) contacting the sample with one or more polypeptides according to any one of claims 20 to 25, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; and (c) detecting the presence or absence of the antibody-polypeptide complex, if any.

2. A method for diagnosing whether a mammal is infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from the mammal with one or more polypeptides according to any one of claims 20 to 25, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; (c) detecting the presence or absence of the antibody-polypeptide complex, if any; and (d) diagnosing the mammal as having Feline lungworm if the antibody-polypeptide complex is present.

3. A method for diagnosing and treating a mammal infected with one or more feline lungworms, the method comprising the steps of: (a) contacting a biological sample from a cat with one or more polypeptides according to any one of claims 20 to 25, wherein the one or more polypeptides are capable of specifically binding to one or more feline lungworm antibodies; (b) forming an antibody-polypeptide complex in the presence of one or more antibodies, if any, in the sample; (c) detecting the presence or absence of the antibody-polypeptide complex, if any; (d) diagnosing the mammal as having feline lungworm if the antibody-polypeptide complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat said mammal having a feline lungworm infection.

4. A method for detecting the presence or absence of one or more feline lungworm antigens in a biological sample, the method comprising: (a) contacting a sample from a cat with one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 20 to 25; (b) forming an antibody-antigen complex in the presence of said one or more feline lungworm antigens, if any, in said sample; and (c) detecting the presence or absence of the antibody-antigen complex, if any.

5. A method for diagnosing whether a mammal is infected with one or more feline lungworms, the method comprising the following steps: (a) contacting a biological sample from the mammal with one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 20 to 25; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if any, in the sample; (c) detecting the presence or absence of the antibody-antigen complex, if any; and (d) if the antibody-antigen complex is present, diagnosing the mammal as having feline lungworm.

6. A method for diagnosing and treating a mammal infected with one or more feline lungworms, the method comprising the steps of: (a) contacting a biological sample from a mammal with one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 20 to 25; (b) forming an antibody-antigen complex in the presence of one or more feline lungworm antigens, if any, in the sample; (c) detecting the presence or absence of the antibody-antigen complex, if any; (d) diagnosing the mammal as having feline lungworm if the antibody-antigen complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat said mammal having a feline lungworm infection.

7. The method according to claim 3 or 6, wherein: Step (e) further comprises one or more additional therapeutic agents to (a) treat an infection caused by one or more helminthic parasites, one or more non-helminthic 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 to (b) control, eliminate or kill intermediate hosts of the feline lungworm parasite, flatworm parasites, helminth parasites, non-helminth parasites, viruses, fungi, protozoa or bacteria.

9. The method according to any one of claims 1 to 8, wherein: The one or more antibodies bind to Aelurostrongylus abstrusus but do not bind to antibodies to Capillaria aerophila or Troglostrongylus brevior.

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

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

12. The method according to claim 10 or 11, wherein: The ascaris 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.

13. The method according to any one of claims 1 to 12, wherein: The step (c) of detecting the presence or absence of the complex further comprises the step of providing at least one second antibody that binds to one or more complexes.

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

15. The method according to claim 14, wherein: The solid support forms part of an ELISA device.

16. The method according to claim 15, wherein: The ELISA device is a lateral flow immunoassay device.

17. The method according to any one of claims 1 to 16, wherein: The mammal is a cat.

18. A polypeptide comprising the amino acid sequence of 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 combination), SEQ ID NO:78 (modified D678), SEQ ID NO:79 (D678-amino extension), SEQ ID NO:81, SEQ ID NO:82 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 XXXWF (SEQ ID NO:117), wherein, (a) retaining amino acids W and F at positions 4 and 5, respectively, and replacing at least one X at positions 1, 2 and 3 with a conservative or non-conservative amino acid; (b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2 and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids or non-conservative amino acids.

19. The polypeptide according to claim 18, wherein The polypeptide is selected from the group consisting of the following 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (D678-amino extension), SEQ ID NO: 81, SEQ ID NO: 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 IDNO: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 XXXWF (SEQ ID NO:117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ; 20. The polypeptide according to claim 18, wherein The polypeptide comprises at least 5 consecutive amino acids of the following amino acid sequence: 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 combination), SEQ ID NO:78 (modified D678), SEQ ID NO:79 (D678-amino extension), SEQ ID NO:81, SEQ ID NO:82 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 (D8M9), SEQ ID NO: 116 (D8M10); or a polypeptide comprising XXXWF (SEQ ID NO: 117), wherein (a) 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 replaced with a conservative or non-conservative amino acid;(b) retaining amino acids W and F at positions 4 and 5, respectively, and performing conservative amino acid substitutions of X at positions 1, 2, and 3; (c) retaining amino acids W and F at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) replacing amino acid W at position 4 or amino acid F at position 5 with a conservative amino acid, wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ; 21. The polypeptide according to claim 18, 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 the full length of TDX1557 amino acids.

22. The polypeptide according to claim 18, wherein The polypeptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids as shown in SEQ ID NO:2-SEQ ID NO:18 and SEQ ID NO:78-SEQ ID NO:

117.

23. The polypeptide according to claim 20, wherein The polypeptide has 10-25 linked amino acids and comprises at least 5, 6, 7, 8, 9 or 10 consecutive amino acids as shown in the following 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 combination), SEQ ID NO: 78 (modified D678), SEQ ID NO: 79 (modified D678), SEQ ID NO: 80 (modified D678), SEQ ID NO: 81 (modified D678), SEQ ID NO: 82 (modified D678), SEQ ID NO: 83 (modified D678), SEQ ID NO: 84 (modified D678), SEQ ID NO: 85 (modified D678), SEQ ID NO: 86 (modified D678), SEQ ID NO: 87 (modified D678), SEQ ID NO: 88 (modified D678), SEQ ID NO: 89 (modified D678), SEQ ID NO: 90 (modified D678), SEQ ID NO: 91 (modified D678), SEQ ID NO: 92 ( NO:79 (d678-amino extension), 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 (D8M9), SEQ ID NO: 116 (D8M10);or a polypeptide comprising XXXWF (SEQ ID NO: 117), wherein (a) amino acids W and F are retained at positions 4 and 5, respectively, and at least one X at positions 1, 2, and 3 is replaced with a conservative or non-conservative amino acid; (b) amino acids W and F are retained at positions 4 and 5, respectively, and conservative amino acid substitutions of X are made at positions 1, 2, and 3; (c) amino acids W and F are retained at positions 4 and 5, respectively, X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids; and (d) amino acid W at position 4 or amino acid F at position 5 is replaced with a conservative amino acid, and wherein X at positions 1 and 3 are independently amino acids S, conservative amino acids, or non-conservative amino acids, and X at position 2 is amino acid K, conservative amino acids, or non-conservative amino acids. ; 24. The polypeptide according to claim 18, wherein The peptide is labeled or attached to a solid support.

25. A polynucleotide encoding the polypeptide according to any one of claims 18-23.

26. The polynucleotide according to claim 25, wherein The polynucleotide is RNA.

27. The polynucleotide according to claim 26, wherein The polynucleotide comprises one or more modified uridine residues.

28. The polynucleotide according to claim 27, wherein The modified uridine residue is pseudouridine or methylpseudouridine.

29. The polynucleotide according to claim 25, wherein The polynucleotide is mRNA.

30. The polynucleotide according to claim 29, wherein The polynucleotide is contained within a lipid nanoparticle.

31. A vaccine composition comprising one or more polynucleotides according to any one of claims 25-30 and a pharmaceutically acceptable carrier.

32. A vaccine composition comprising one or more polypeptides according to any one of claims 18-24 and a pharmaceutically acceptable carrier.

33. An immune complex comprising one or more polypeptides according to any one of claims 18-24 and one or more feline lungworm antibodies that specifically bind to the one or more polypeptides.

34. An immune complex comprising one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 18-24 and one or more feline lungworm antigens that specifically bind to the one or more antibodies.

35. A device for detecting the presence or absence of one or more feline lungworm antibodies from a biological sample, the device comprising a solid support, wherein: The solid support has immobilized thereon one or more polypeptides according to any one of claims 18-24.

36. The device according to claim 34, wherein The one or more polypeptides do not specifically cross-react with one or more antibodies to Ascaris lumbricoides, Tapeworms, Hookworms, Capillaria aeruginosa, or Brachystenoma strongyloides.

37. The device according to claim 35, wherein: The ascaris is of the genus Toxocara; and / or the tapeworm is of the genus Taenia or Dipylidium; and / or the hookworm is of the genus Ancylostoma.

38. A device for detecting the presence or absence of one or more feline lungworm antigens from a biological sample, the device comprising a solid support, wherein: The solid support has immobilized thereon one or more antibodies that specifically bind to one or more polypeptides according to any one of claims 18-24.

39. The device according to claim 38, wherein The one or more antibodies do not specifically cross-react with one or more antigens of Ascaris lumbricoides, Tapeworms, Hookworms, Capillaria aeruginosa, or Brachystenus strongyloides.

40. The device according to claim 39, wherein The ascaris is of the genus Toxocara; and / or the tapeworm is of the genus Taenia or Dipylidium; and / or the hookworm is of the genus Ancylostoma.

41. A kit comprising a device according to any one of claims 35-37 or a device according to any one of claims 38-40.

42. A method of immunizing a mammal against feline lungworm, the method comprising administering the vaccine composition of claim 31 or 32.

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