Methods of reducing intrauterine transmission of salmonella dublin infection

Abstract

Methods of reducing or preventing intrauterine transmission of Salmonella Dublin in a subject, typically a cow, are provided. The methods include a first administration, and optionally second or more administrations, to a pregnant cow of an effective amount of an immunogenic composition to induce an immune response against S. Dublin in the cow. In some forms, the cow is infected with S. Dublin. In some forms, two or more cows are treated in parallel, for example, all cows that inseminated or otherwise became pregnant in parallel. Typically, the first administration, and optionally second or more administrations, are administered to the cow at a time(s) that reduces intrauterine transmission of S. Dublin infection from the cow to one or more of her calves compared to unvaccinated control cows and / or cows administered according to a different administration regimen. Exemplary methods of administration are provided.

Description

[0001] METHODS OF REDUCING INTRAUTERINE TRANSMISSION OF SALMONELLA DUBLIN INFECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application 63 / 675,639 filed on July 25, 2024; which is incorporated by reference herein, in its entirety.

[0004] REFERENCE TO THE SEQUENCE LISTING

[0005] The Sequence Listing submitted as an XML file named “BIAH_PM12035_PCT_ST26.xml,” created on July 23, 2025, and having a size of 4,593 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0006] FIELD OF THE INVENTION

[0007] The invention is generally directed to the field of immunization methods, particularly in agricultural animals.

[0008] BACKGROUND OF THE INVENTION

[0009] Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is a bovine host- adapted serotype with an increasing prevalence of infections in dairy cattle in the US and Canada has increased over the past years (Cummings et al., 2009; 2010; NAHMS, 2014; Perry et al., 2023). S. Dublin is the most commonly isolated serovar in several veterinary diagnostic laboratories (Erdman et al., 2012; Valenzuela et al., 2017; Holschbach and Peek, 2018). Furthermore, most US isolates of 5. Dublin are multi-drug antimicrobial resistant (Erdman et al., 2012; Valenzuela et al., 2017; Holschbach and Peek, 2018), although with reasonable susceptibility to enrofloxacin (Velasquez-Munoz et al., 2024). However, this antimicrobial is not labeled as a treatment for S. Dublin infections, and extra-label use of fluoroquinolones such as enrofloxacin is prohibited for food animals in the US (FDA, 1994). Thus, no effective antimicrobial is currently available to treat S. Dublin infections in cattle in the US. Also, S. Dublin is a public health concern as it causes fatal multi-drug resistant infections in humans via contaminated foods or contact with infected animals (Harvey et al., 2017, CDC, 2019, Paudyal et al., 2019), with mortality in humans being 4.4 times higher than in other Salmonella infections (Helms et al., 2003). Therefore, establishing reliable control strategies for S. Dublin is important to the dairy industry.

[0010] Infection with Salmonella host-adapted serovar Dublin can occur in utero or when calves ingest the bacteria shed by infected cows or calves (Richardson, 1973). After colonizing the gastrointestinal tract, S. Dublin can spread through the bloodstream into organs such as the liver, spleen, lung, or lymph nodes. Calves that survive the infection have a high probability of becoming asymptomatic carriers for life (Nielsen et al., 2004), shedding low numbers of bacteria in their secretions for years (Nielsen, 2013a). This potential for asymptomatic infection and latent carriers of disease creates important challenges in managing Salmonella spp. transmission (Costa et al., 2012). Latent carrier cows infect calves in utero and shed bacteria during calving (Counter and Gibson, 1980; Nielsen et al., 2004; Hanson et al., 2016). Thus, controlling the spread of S. Dublin from latent carriers to newborns is needed to reduce disease transmission in affected herds (Velasquez-Munoz et al., 2024).

[0011] It is therefore an object of the invention to provide compositions and means of reducing .S'. Dublin disease transmission.

[0012] SUMMARY OF THE INVENTION

[0013] The studies below report the discovery that intrauterine transmission is an important route of transmission, perhaps the most important route of transmission, of S. Dublin infection from cows to their calves. Thus, methods of reducing or preventing intrauterine transmission of Salmonella Dublin in a subject, typically a cow, are provided. The methods typically include a first administration, and optionally second or more administrations, to a pregnant cow of an effective amount of an immunogenic composition to induce an immune response against S. Dublin in the cow.

[0014] In some forms, the cow is infected with S. Dublin. The cow can be a latent carrier of S. Dublin. In some forms, the methods include detecting S. Dublin bacteria and / or antibodies thereto in the cow. In some forms, the cow is not infected with S. Dublin. In some forms, two or more cows are treated in parallel, for example, all cows that inseminated or otherwise became pregnant in parallel. Such cows can be a mix of infected and uninfected cows.

[0015] Typically, the first administration, and optionally second or more administrations, are administered to the cow at a time(s) that reduces intrauterine transmission of 5. Dublin infection from the cow to one or more of her calves compared to unvaccinated control cows and / or cows administered according to a different administration regimen.

[0016] In some forms, the first administration is made after or coincident with confirmation of pregnancy.

[0017] In some forms, the first administration is made in the range of 120 days or 250 days, inclusive, post-conception, or any integer subrange or specific day therebetween. For example, the first administration can be in the range of 120 days or 180 days, inclusive, post-conception, or any integer subrange or specific day therebetween. In some forms, the cow is within 5-10 days of dry-off. Thus, in some forms, the cow is 200- 250 days, optionally 215-226 days, post-conception, or any integer subrange or specific day therebetween.

[0018] In some forms, the second administration is 10-31, optionally 12-28, optionally 12, 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the first administration.

[0019] In some forms, the second administration is between 155 and 287 days (i.e.. 5-9 months), or between 155-218 days (i.e., 5-7 months) post-conception or any integer subrange or specific day therebetween.

[0020] In some forms, the subject is administered the composition subcutaneously, although other routes of administration are also contemplated and provided.

[0021] The immunogenic composition typically includes an antigen derived from a Salmonella, preferably S. Dublin. For example, the immunogenic composition can include an .S'. Dublin bacteria or lysate or bactrin formed therefrom. In some forms, the .S'. Dublin bacteria is attenuated, inactivated, and / or avirulent. In a particular form, the immunogenic composition includes or is ENTER VENE®-d; Boehringer Ingelheim Animal Health. Any of the methods can include one or more additional steps or means of reducing or preventing S. Dublin infection in the cow, or her herd.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a diagram representing the number of animals included at each stage of the study and reasons for their removal.

[0024] Figure 2 is a bar graph showing the number of milk tests testing positive for Salmonella Dublin antibodies in each farm. Results are expressed as the number (%) of cows with positive milk antibody ELISA tests. Milk samples were collected every other month for a total of 3 consecutive tests. Farm B had 10 (3.6%), 6 (2.2%), and 3 (1.0%) cows with one, two, and three positive tests, respectively. Cows with all 3 positive tests were considered S. Dublin latent carriers.

[0025] Figure 3 is a dot plot showing comparison of sera Salmonella Dublin antibodies at birth between calves bom to Vaccine (n=60) and Control (n=58) cows. Results are expressed as the % positivity of the ELISA. Samples with a negative % positivity result were assigned a value of 0. Results of % positivity > 35% are indicative of antibodies present in the sample, and the greater % positivity is indicative of a greater antibody concentration. The results were compared statistically with a two-tailed Mann-Whitney U test. DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Definitions

[0027] The term "immunogenic composition" refers to any composition able, once it has been introduced into an animal, to induce or stimulate an immune response against Salmonella Dublin.

[0028] The term "vaccine composition" or "vaccine" refers to any composition able, once it has been introduced into an animal, to induce or stimulate a protective immune response against diseases caused by Salmonella Dublin and / or to induce or stimulate a protective immune response to prevent or to reduce the carriage of Salmonella Dublin in animals.

[0029] As used herein, the terms “effective amount” or “therapeutically effective amount” means a dosage sufficient to provide treatment for a disorder, disease, or condition being treated, to induce or enhance an immune response, or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subjectdependent variables (e.g., age, immune system health, etc.), the disease, the disease stage, and the treatment being effected.

[0030] The terms “patient” or “subject” to be treated and / or used in accordance with any of the aspect as described herein refers to animals, most typically cattle, particularly cows.

[0031] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0032] As used herein, the term “pharmaceutically-acceptable carrier” refers to an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. It can further mean one or more compatible solid or liquid fillers, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal.

[0033] As used herein, the term “treating” includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with a disease or disorder.

[0034] As used herein, the term “reduce”, “inhibit”, “alleviate” or “decrease” are used relative to a control. One of skill in the art would readily identify the appropriate control to use for each experiment.

[0035] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0036] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0037] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0038] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0039] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0040] II. Methods of Inducing an Immune Response

[0041] Methods of inducing immune response in a subject are provided.

[0042] The disclosed methods typically include administering the subject an effective amount of an immunogenic composition or vaccine to induce an immune response in the subject against S. Dublin. Suitable compositions are discussed in more detail below.

[0043] An immune response to a composition or vaccine is the development in the host of a cellular and / or antibody -mediated immune response to the composition or vaccine of interest. Usually, such a response includes the subject producing antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

[0044] By vaccine is meant an agent used to stimulate the immune system of an individual so that protection is provided against an antigen not recognized as a self-antigen by the immune system. Immunization refers to the process of inducing a continuing high level of antibody and / or cellular immune response in which T-lymphocytes can either kill the invading microbe and / or activate other cells (e.g., phagocytes) to do so in an individual, which is directed against a microbe or antigen to which the organism has been previously exposed. The phrase "immune system" is intended to refer to the anatomical features and mechanisms by which an individual produces antibodies against an antigenic material that invades the cells of the individual or the extra-cellular fluid of the individual and is also intended to include cellular immune responses. In the case of antibody production, the antibody so produced can belong to any of the immunological classes, such as immunoglobulins A, D, E, G or M. Of particular interest are vaccines which stimulate production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm- blooded animals, although vaccines of the invention are not limited to those which stimulate IgA production. For example, vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example cellular and humoral immunity. Immune responses to-antigens-are well studied and widely reported. A survey of immunology is provided in Elgert, Klaus D., Immunology, Wiley Liss, Inc., (1996); Stites et al., Basic & Clinical Immunology; 7th Ed., Appleton & Lange, (1991) the entirety of which are incorporated herein by reference. A. Subjects

[0045] The subject is typically a mammal, most typically cattle. The subject is typically female, e.g., a cow or heifer. Exemplary, non-limiting breeds include, but are not limited to, Black Angus, Belted Galloway, Brahman, Charolais, Dexter, Gelbvieh, Hereford, Holstein, Limousin, Piedmontese, Red Angus, Scottish Highland, Shorthorn, Simmental, Texas Longhorn, and Watusi.

[0046] The subject is typically pregnant.

[0047] The subject can be dry at the outset or during part or all of the provided methods. A dry cow refers to a dairy cow that is in a stage of their lactation cycle where milk production ceases prior to calving. This part of the lactation cycle is referred to as the cow’s dry period and typically last between 40 and 65 days. Dry cows are typically divided into two groups: far-off (60-21 days before calving) and close-up (21 days to calving). Once the cow has entered this stage, producers may seal the cow’s teat. Teat sealants may be internally or externally administered depending on the chosen protocol. Antibiotics are commonly used within treatments, which will be inserted into the teat before the sealant is applied. Once these treatments are administered, a post-milking teat dip or spray may be applied. This dry period is a part of the lactation cycle and is important for the cow’s health, the newborn calf and future milk production, as it allows the cow time to rest, eat and prepare for the onset of a new lactation. The cow synthetizes colostrum for the newly bom calf in the last weeks of the dry period.

[0048] Thus, in some forms, the disclosed methods include administering a subject, typically a dry subject, typically a dry cow, an effective amount of the immunogenic composition or vaccine. In some forms, the subject has a sealed teat.

[0049] In some forms, the subject is a carrier of Salmonella Dublin. In some forms, the subject is a latent carrier. Latent carriers of S. Dublin are animals that are infected but are apparently healthy most of the time and only infectious at other times, with the ability of changing back and forth from one to the other. This is different from a more typical, generic latency period of other pathogens, which is only the symptom-free pre-infectious period. The importance of latent carriers in the epidemiology of S. Dublin is important as they are defined as animals with a persistent infection without clinical signs but with an intermittent shedding of the pathogen to the environment through feces or secretions. Thus, in some forms, the subject is asymptomatic for S. Dublin infection. Latent carriers may also be clinically ill or reactivate the shedding when the immune system is compromised.

[0050] In some forms, the subject is at risk of developing a new S. Dublin infection. There is an association between age and the productive stage of the cattle with the risk of new infections and latent carrier status. For example, calves younger than 12 weeks of age are highly susceptible to infection of S. Dublin. Repeated infection in young calves leads to a high proportion of resistant adult cattle and an increase in abortions in the last third of gestation. Moreover, it was reported that heifers infected between 1 year of life and calving were 11 times more likely to become a latent carrier than cows infected in mid or late lactation. A similar situation was described for cows infected in the peripartum, as they were 4 times more likely to become latent carriers. Additionally, stress is an important factor associated with the infection of susceptible animals and the shedding of S. Dublin in latent carriers. Calving and transportation have been identified as periods of increased stress and shedding of S. Dublin. Furthermore, any management practice that may increase the stress of the animals and the direct contact with infected animals that are actively shedding S. Dublin may result in infection of susceptible animals or outbreaks of disease.

[0051] 1. Carrier Detection

[0052] In some forms, the subject is identified as infected with S. Dublin prior to treatment. There are two main approaches for the diagnosis of S. Dublin: the detection of bacteria and the detection of antibodies. They can be performed in individual animals or the herd. See, e.g., Velasquez- Munoz, el al., Front Vet Sci. 2024 Jan 9:10:1331767. doi: 10.3389 / fvets.2023.1331767. eCollection 2023, and Nielsen, Vet Microbiol. (2013) 162: 1-9. 10.1016 / j.vetmic.2012.08.003, each of which is specifically incorporated by reference herein in its entirety. Each of these techniques are exemplified in the experiments below. a. Detection of bacteria

[0053] Bacteriological culture has been useful for isolating and identifying S. Dublin to trace infections and active shedders. Bacteriological culture can be performed utilizing a variety of samples, including feces and fluids from live animals, organs from necropsies, aborted fetuses, or environmental samples. This method aims to isolate live bacteria. Thus, the procedure involves a pre-enrichment and a selective enrichment to allow bacterial growth, followed by plating and confirmation. This method has been described as more useful in acute infections and clinically ill animals, as the correct isolation will depend on the number of bacteria in the sample. For that reason, the sensitivity of this assay has been described as low, and it has a limitation that latent carriers might be undetected due to the intermittent and low bacterial load of fecal shedding of S. Dublin. Bacteriological culture can be prepared using samples from, e.g., manure pits, drinking water, milk filters, and feces

[0054] A potentially more sensitive and faster method for the detection of genetic material of Salmonella is molecular techniques such as sequencing and / or polymerase chain reaction (PCR) such as real-time PCR. See, e.g., Persson et al., J Appl Microbiol. (2012) 113:615-21. 10.1111 / j.1365-2672.2012.05378.x, and Goodman, et al. J Vet Diagn Invest. (2017) 29:844-51. 10.1177 / 1040638717728315, which are specifically incorporated by reference in their entireties, and the experiments below which described S. Dublin-specific PCR assays. In some forms, this procedure includes a pre-enrichment of the sample from lysates or extracted DNA. To increase sensitivity, a DNA extraction can be use. It is believed a multiplex PCR can be used to discriminate Salmonella Enteritidis, Salmonella Poliorum, and S. Dublin (109). This assay is based on detecting three genes (tcpS, lygD, and flhB), where tcpS exists only in the serovars mentioned. b. Detection of antibodies

[0055] The detection of immunoglobulins against S. Dublin can be performed through an Enzyme- linked immunosorbent assay (ELISA). This method has a lower cost than bacteriological culture, and it can be used as a monitoring strategy in the herd to identify latent carriers during programs of control and eradication. Salmonella Dublin is part of the D-serogroup of Salmonella and has the antigenic factors 01, 09, and 012; therefore, cross-reaction between serovars sharing O antigens may occur. The ELISA is typically based on detecting immunoglobulins directed to the LPS O- antigen from serum, milk, and bulk tank milk (BTM) samples. A kit is commercially available in several countries for monitoring and surveillance of Salmonella infections in cattle herds (Applied Biosystems™, Massachusetts, USA). The results provided in this ELISA are typically semi- quantitative for antibody concentration as they are expressed in 0DC% (optical density coefficient). The interpretation of the result is based on an estimated cut-off point to determine positive animals depending on the sample. The 0DC% cut-off for serum, milk from an individual, or BTM is 35 0DC%. A positive correlation exists between the 0DC% and antibody concentration in a sample. In BTM, the greater the ODC%, the higher the spread of infection in the herd.

[0056] To identify latent carriers and the intermittent shedding of S. Dublin, sequential samples can be obtained from individual animals by using milk or serum samples. For example, studies from countries with eradication plans recommend that cows be sampled quarterly.

[0057] B. Methods of Administration

[0058] Typically, the method includes at least one administration of an immunogenic composition or vaccine, to cow, typically a pregnant cow.

[0059] The studies below report the discovery that intrauterine transmission is an important route of transmission, perhaps the most important route of transmission, of S. Dublin infection from cows to their calves. Thus, an aim of the disclosed methods is to reduce intrauterine transmission of .S'. Dublin to calves of the pregnant cows. Therefore, in some forms, the structure the one, two, or more administrations of the immunogenic composition or vaccine are designed to induce, and optionally maximize S. Dublin immunity in the calves at a time when they are susceptible, optionally maximally susceptible, to intrauterine S. Dublin infection. Gestation length for cattle ranges from 279 to 287 days. In some forms, an administration is made after pregnancy is confirmed. In some forms, an administration is made to a pregnant cow in the range of 120 days or 180 days, inclusive, post-conception, or any integer subrange or specific day therebetween, all of which are thus expressly disclosed. In some forms, the foregoing administrations are the first (i.e., “prime”) of two or more administrations.

[0060] It is often also desirable to administer one or more booster applications of the composition or vaccine sometime after the initial (i.e., prime) administration. Such booster applications can be administered at any time after the first administration that has beneficial effects of the vaccine. The second and optionally further administrations are also during the pregnancy period. In some forms, a first booster application is applied between 5 and 35 days, between 12 and 30 days, or between 12 and 28, post-conception or any integer subrange or specific day therebetween, all of which are thus expressly disclosed. For example, in some form a booster is administered 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days after the first, prime administration.

[0061] In some forms, the last administration is during between 155 and 287 days inclusive i.e., 5- 9 months), or between 155 and 218 days inclusive (i.e., 5-7 months) post-conception or any integer subrange or specific day therebetween, all of which are thus expressly disclosed. Additionally or alternatively, in some forms, the first administration is during between 155 and 287 days inclusive (i.e., 5-9 months), or between 155 and 218 inclusive (i.e., 5-7 months) post-conception or any integer subrange or specific day therebetween, all of which are thus expressly disclosed.

[0062] In some forms, one or more of the administrations occur during the cow’s dry period. In a particular form, a booster administration is made at the time of, or just prior to, or just after (e.g., 1, 6, 5, 4, 3, 2, or 1 day(s)) the initiation of the dry off period. The prime administration can be a suitable number of days before the booster administration, such as those mentioned above. For example, in some forms, the prime administration is 5-30 days before the initiation of the dry off period, or any integer subrange or specific day therebetween, all of which are thus expressly disclosed.

[0063] In some forms, two or more cows are treated in parallel. For example, in some forms, all of the pregnancy-positive cows that were inseminated in parallel are also vaccinated in parallel.

[0064] In some forms, the administration protocol is repeated for the same cow(s) each time she / they become pregnant.

[0065] Suitable routes of administration of immunogenic compositions or vaccines according to the methods of the present disclosure for prime- and / or boost- administration for non-respiratory diseases, such as diseases of the tissues, circulatory system or gut, is usually by subcutaneous or intramuscular injection, or by oral administration, either by inoculation or by application in drinking water. However, other methods of administration are also contemplated and include inhalation (e.g., in an aerosol or spray) to thereby contact the mucosal surfaces of the upper respiratory tract. An immunogenic composition or vaccine can be administered by a syringe with a needle or by a needle-free apparatus (such as, for example, Pigjet, Avijet, Dermojet, Vitajet or Biojector (Bioject, Oregon, USA), see US Published Application No. 2006 / 0034867).

[0066] Doses of immunogenic compositions or vaccines for administration according to the methods can include from about 0.1 mL to about 5.0 mL, preferably from about 1.0 mL to about 3.0 mL, and more preferably from about 1.5 mL to about 2.5 mL. These doses have from about 106colony forming unit per dose (CFU / dose) to about 1010CFU / dose of a Salmonella strain, or any integer subrange or specific dose therebetween, each of which is expressly disclosed. In particular forms, the dose is 107- 109CFU, optionally about 108CFU, optionally such as about 4.9 x 107CFU / mL in 2 ml.

[0067] III. Compositions

[0068] A. Antigens

[0069] The immunogenic compositions and vaccines typically include an effective amount of antigen and optionally an adjuvant, in a pharmaceutically acceptable carrier. The antigen can be live or inactivated bacterium, or a lysate thereof. Antigens can induce a protective immune response against more than one Salmonella serovar and / or against Salmonella of more than one Kauffmann- White classification group. Thus, although the antigen is typically derived from S. Dublin, or is an 5. Dublin bacterium, other antigens, including other bacteria, are also contemplated provided they provide an (e.g., cross-reactive) immune response against .S'. Dublin.

[0070] The Salmonella can be avirulent. Reference to avirulence is intended to mean that a particular virulent microbe strain has been modified so that it is incapable of inducing a full suite of symptoms of the disease state that is normally associated with its virulent pathogenic counterpart. Thus, avirulence includes a state of diminished virulence or ability to produce disease conditions and the avirulent microorganisms are not necessarily completely absent of any ability to impair normal physiological functioning of the host. In addition, an avirulent microbe is not necessarily incapable of ever functioning as a pathogen, but the particular microbe being used is avirulent with respect to the particular individual being treated.

[0071] Salmonella species can be rationally attenuated by introducing non-reverting defined mutations into the genome to produce live vaccine strains. Several genes have been identified, which when mutated, will attenuate Salmonellae. In particular, salmonella strains harboring nonreverting mutations in genes involved in the pre-chorismate biosynthetic pathway make excellent oral vaccines evoking strong humoral, local and cellular immune responses in the host (Chatfield S. N. et al., Vaccine, 1989, 7(6): 495-8; Chatfield S. N. et al., FEMS Immunol. Mecl. Microbiol., 1993, 7(1): 1-7), in aro genes of the aromatic biosynthetic pathway (EP-B 1-0322237), in the transcriptional regulator RfaH mutants of Salmonella Typhimurium, which are efficient as attenuated oral vaccines against salmonellosis in mice (Nagy G. et al., Infect. Immun., 2006, 74(10): 5914-25).

[0072] Salmonella may be attenuated by modification of the genome structure of the bacteria, such as by deletion of part of a Salmonella gene, by insertion of heterologous nucleotide sequence into a Salmonella gene, and / or by substitution of part of a Salmonella gene by heterologous nucleotide sequence. It is possible to attenuate Salmonella by introducing mutations that (i) confer auxotrophy, (ii) interfere with sugar metabolism and Lipopolysaccharides biosynthesis or (iii) affect some global means of regulating genes needed for a full display of virulence.

[0073] For example, attenuated Salmonella can be bacteria having at least one streptomycin and one rifampicin resistance metabolism drift mutation for the attenuation (EP-B 1-0642796); attenuated by a first mutation in the phoP regulatory region causing constitutive expression of a gene under the control of said region and by a second mutation at a pag or prg gene (EP-B 1- 0563311); attenuated by a non-reverting mutation in the htrA gene (U.S. Pat. No. 5,804,194); attenuated Salmonella that exhibits auxotrophy to one or more growth factors selected from the group including phenylalanine, tyrosine, tryptophan and para-aminobenzoic acid, such that it is incapable of growing on a minimal medium in the absence of said one or more growth factors (U.S. Pat. No. 6,231,871); auxotrophic mutants derived through N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, (Martin G. et al., Berl. Munch. Tierarztl. Wschr., 1996, 109(10): 325-9); attenuated Salmonella harboring a non-reverting mutation in each of two discrete aro genes of the aromatic biosynthetic pathway, such as aroA and aroC, aroA and aroD, aroA and aroE (EP-B 1-0322237); attenuated Salmonella genetically-defined aroC derivative (LVR02 strain, see Betancor L. et al., Vet. Microbiol., 2005, 107(1-2): 81-9); and attenuated Salmonella having a mutation that inactivates a gene selected from: hupA, dksA, rfaY, sipC or clpB (W0-A1 -98 / 02523). Attenuating mutations can also be obtained by insertion of a transposon.

[0074] The Salmonella may be chemically inactivated by treatment with inactivating agents, such as formaldehyde, ethyleneimine, ethyleneimine amide derivatives (for example acetylethyleneimine), propyleneimine, |3-propiolactone, thimerosal, acetone or heat-inactivation. In a preferred form, the inactivating agent is formaldehyde. The antigen can be a bacterin.

[0075] See also, e.g., U.S. Pat. Nos. 7,045,122; 6,923,957; 6,905,691 ; 6,605,285; 5,843,426; 5,733,760; 5,424,065; 5,389,368; 5,387,744; 5,468,485; 5,294,441; 6,592,869; and 8,062,645, which relate to Salmonella vaccines, including avirulent, attenuated, and inactivated vaccines.

[0076] Attenuated Salmonella vaccines and inactivated Salmonella vaccines are commercially available. In a particularly preferred form, the immunogenic composition or vaccine is the Salmonella Dublin vaccine ENTER VENE®-d (Boehringer Ingelheim). In a more specific forms, vaccinated cows receive 2 mL of a commercial live culture S. Dublin vaccine (ENTER VENE®-d; Boehringer Ingelheim Animal Health) subcutaneously (SC) optionally within 7 d of dry-off (e.g., 215 to 226 actual days pregnant), and a booster 12-30 days, optionally 12, 13, 14, or 28 days after the primary vaccination. In other forms, the vaccine is a bacterin, such as Salmonella Dublin- Typhimurium Bacterin (e.g., Colorado Serum Company, catalog no. 11602).

[0077] B. Adjuvants

[0078] Adjuvant(s) can be added to the antigenic composition, e.g., a bacterial suspension, notably obtained after culture and inactivation. An adjuvant may be chosen from aluminum hydroxide, saponin, any water-in-oil emulsion or oil-in-water emulsion (see e.g., Herbert W. J., The Lancet, 1965, Oct. 16: 771 ; Brugh M. et al., Am. J. Vet. Res., 1983, 44(1): 72-5; Boersma W. J. A. et al., 44.sup.th Forum in Immunology, "Characteristics and use of new-generation adjuvants", 503-511; Gast et al., Avian Diseases, 1993, 37 (4): 1085-91 ; Stone, Avian Diseases, 1993, 37: 399-405; Stone et al., Avian Diseases, 1990, 34: 979-983; Stone et al., Avian Diseases, 1983, 27(3): 688-697; U.S. Pat. No. 3,919,411; WO-A-05 / 009462, all herein incorporated by reference).

[0079] Examples of adjuvants include, but are not limited to, oil-in-water, water-in-oil-in-water emulsions based on mineral oil and / or vegetable oil and non-ionic surfactants such as block copolymers, TWEEN® (polysorbate), SPAN® (sorbitan monoester). Such emulsions are notably those described in page 147 of "Vaccine Design-The Subunit and Adjuvant Approach", Pharmaceutical Biotechnology, 1995, volume 6, edited by Michael F. Powell and Mark I. Newman, Plenum Press, New York and London, or TS emulsions, notably the TS6 emulsion, and LF emulsions, notably LF2 emulsion (for both TS and LF emulsions, see WO-A-04 / 024027). Other suitable adjuvants are, for example, vitamin E, saponins, and polymers of crosslinked acrylic or methacrylic acid, i.e., CARBOPOL® (carbomer) (Noveon; see WO-A-99 / 51269; WO-A- 99 / 44633), HAVLOGEN® (carbomer), aluminium hydroxide or aluminium phosphate ("Vaccine Design, The subunit and adjuvant approach", Pharmaceutical Biotechnology, vol. 6, Edited by Michael F. Powell and Mark J. Newman, 1995, Plenum Press New York), biological adjuvants (e.g., C4b, notably murine C4b (Ogata R T et al., J. Biol. Chem. 1989, 264(28): 16565-16572) or equine C4b, GM-CSF, notably equine GM-CSF (U.S. Pat. No. 6,645,740)), toxins (e.g., cholera toxins CTA or CTB, Escherichia coli heat-labile toxins LTA or LTB (Olsen C W et al., Vaccine, 1997, 15(10): 1149-1156; Fingerut E et al., Vaccine, 2005, 23(38): 4685-4696; Zurbriggen R et al., Expert Rev Vaccines, 2003, 2(2): 295-304; Peppoloni S et al., Expert Rev Vaccines, 2003, 2(2): 285-293)), and CpG (e.g., CpG #2395 (see Jurk M et al., Immunobiology 2004, 209(1-2): 141-154), CpG #2142 (see SEQ. ID. NO: 890 in EP-B1-1,221,955), CpG #2135, CpG #2007, CpG #2336). Polymers of crosslinked acrylic or methacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or poly alcohols are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g., vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. Products sold under the name CARBOPOL® (carbomer) are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to CARBOPOL® (carbomer) 974P, 934P, 934, 940 and 97 IP.

[0080] The immunogenic compositions and vaccines can be freeze-dried advantageously with a stabilizer. Freeze-drying can be done according to well-known standard freeze-drying procedures. The pharmaceutically or veterinarily acceptable stabilizers may be carbohydrates (e.g., sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al., Cryobiology 1983, 20(3): 318-23; Israeli E etal., Cryobiology 1993, 30(5): 519-23), proteins such as peptone, albumin, lactalbumin or casein, protein containing agents such as skimmed milk (Mills C K et al., Cryobiology 1988, 25(2): 148-52; Wolff E et al., Cryobiology 1990, 27(5): 569-75), and buffers (e.g. phosphate buffer, alkaline metal phosphate buffer). An adjuvant may be used to make soluble the freeze-dried preparations.

[0081] Examples of useful oils include, but are not limited to, mineral oil, such as paraffin oil, DRAKEOL® (mineral oil) 6VR, MARCOL® 80(mineral oil); MARCOL® 52(mineral oil); terpene oils such as squalene and squalane; vegetable oils such as soybean oil, olive oil, com oil, jojoba oil, peanut oil, cotton-seed oil, sunflower oil, safflower oil, sesame oil, apricot oil, avocado oil, wheat germ oil, canola oil, Linseed oil, and almond oil; fish oils such as shark oil, orange roughy oil, Menhaden oil, and cod liver oil; animal oils such as mink oil, lard oil, and chicken fat oil.

[0082] Examples of surfactants used in emulsion vaccines include ARLACEL® 80 (sorbitan monooleate), TWEEN® 80 (Polysorbate 80), SPAN® 80 (Sorbitan monooleate), SPAN® 85 (Sorbitan trioleate), ARLACEL® 83 (sorbitan sesquioleate), ARLACEL® 85 (sorbitan sesquioleate), and TWEEN® 61 (polyoxyethylene sorbitan), for example. Surfactants suitable for animal and vegetable water-in-oil vaccines include crude yellow, and purified beeswax, for example. Furthermore, surfactants suitable for vaccines containing squalene and squalane include ARLACEL® (sorbitan sesquioleate) and TWEEN® 80 (Polysorbate 80).

[0083] The adjuvant can include an oil to form a water-in-oil emulsion including a paraffin oil and surfactants, notably a paraffin oil, a polyol and fatty acid ester, and an ethoxylated polyol and fatty acid ester.

[0084] C. Methods of Making

[0085] The immunogenic composition or vaccine can be prepared by growing the vaccine strain in suitable growth media and then used as is or formed into a vaccine composition by combining the growing culture, or the cells therefrom, with a suitable diluent. Suitable diluents are preferably liquids and are more preferably a liquid that does not adversely affect the stability and vitality of the vaccine culture and which has a viscosity similar to water so that it will easily form droplets of a coarse spray. The diluent is preferably free of chlorine, antibiotics, antimicrobials, or any other agent that may be harmful to the live vaccine organisms. Vaccine should be dispersible in the diluent so that no solid lumps or chunks of vaccine remain and the diluent should be at a temperature that is not harmful to the live vaccine microbes. Examples of suitable diluents include water, distilled water, de-ionized water, skim milk, water containing Marek's vaccine stabilizer, buffered saline with gelatin, and similar compositions that are well-known to persons of skill in the art. The vaccine is preferably introduced into the diluent while the diluent is at a temperature of approximately room temperature or cooler, more preferably from about 34°C. to about 15°C.

[0086] IV. Combination Treatments

[0087] The disclosed immunogenic compositions and vaccines can be used alone or in combination with other therapies and mitigation techniques commonly used to reduce and / or prevent 5. Dublin infection. In some forms, the subject is treated with an antimicrobial. Exemplary antimicrobials include, but are not limited to, gentamicin, amikacin, cefoxitin, cephalothin, enrofloxacin, meropenem, azithromycin, ceftiofur, danofloxacin, enrofloxacin, florfenicol, trimethoprim / sulfamethoxazole, and tulathromycin.

[0088] Treatment can also include supportive therapy such as correcting the electrolyte imbalance, dehydration, and management of inflammation Electrolyte therapy can be provided orally or intravenously.

[0089] Treatment can include administration of non-steroidal anti-inflammatory drugs to manage inflammation. Strategies for prevention and control of .S'. Dublin can also include cleaning and disinfection of the environment. The first step in decontaminating the environment and equipment is removing organic material ( / .<?., food, manure, bedding), as this can inactivate disinfectants. Secondly, the surfaces can be rinsed with water and a detergent applied. A thorough rinse with water should follow this step. The final step can be the application of a disinfectant in the concentration and contact time described on the label, as any variation in the use of a disinfectant may affect its effectiveness. Salmonella is susceptible to most disinfectants if steps one and two have been appropriately performed.

[0090] Besides cleaning and disinfection, improvement in management practices, such as cleaning water troughs with chlorinated disinfectant twice a week, replacing bedding weekly, and not recycling the water used for flushing pens, have been associated with a decrease in Salmonella incidence in a herd experiencing an outbreak. The sanitation of calving areas should be performed according to their use. Trailers used for the transport of animals should be cleaned and disinfected consistently. Pens should be kept empty for e.g., 2 days before new animals are housed in them. Cleaning and disinfection should be a priority for all equipment used to manage sick calves or to feed calves, including tools used to harvest, store, and provide colostrum and milk, such as esophageal tubes, nipples, bottles, and buckets. In addition, buckets used to feed water and starter in pre- weaned calves should be cleaned daily and positioned to reduce the risk of fecal contamination.

[0091] Even though feeding waste milk can be cost-effective, the provision of raw milk may increase calf morbidity and mortality due to the ingestion of pathogens. Therefore, another hygiene strategy to prevent and control Salmonella infection in pre-weaned calves is the pasteurization of colostrum and milk as it has been shown to reduce microbial populations including S. enterica species.

[0092] Maintaining an adequate stocking density in mature cow pens (i.e., close-up, maternity, fresh cow), heifer pens, and pre-weaned calves housed in groups is a practice that can reduce the contact between animals, the contamination of the environment, and new infections. Overcrowding should be avoided. In addition, the calving area should not be used to house sick animals due to the risk of environmental contamination and infection of newborn calves. Young calves should not have access to or contact with older animals; therefore, strict age group housing in conjunction with adequate stocking density has been recommended to prevent and control 5. Dublin. Animals exhibiting clinical signs of S. Dublin infection should be isolated, and strict cleaning procedures should be in place.

[0093] The management of the newborn and the calving area is important to prevent S. Dublin infections. Some consideration should be taken as latent carriers can reactivate the shedding of bacteria around calving. Newborn calves should be separated from the dam as soon as possible after birth to avoid oral infection due to consumption of colostrum or feces from the dam or other adult cows. Calves in endemic herds should be provided with pasteurized colostrum. Having fewer personnel in charge of calving and colostrum handling can also help prevent S. Dublin infections.

[0094] Avoiding the purchase of cattle from test-positive herds or herds with unknown infection status should be a consistent practice in dairy facilities to prevent the introduction of .S'. Dublin in the herd. Similarly, pasture-based operations should prevent close contact with cattle from neighboring farms, as the introduction of S. Dublin can occur through direct contact.

[0095] Training should be provided to personnel working with animals regarding the risk of zoonotic diseases and their prevention. Particular attention should be put on personnel handling animals during an outbreak of .S'. Dublin or personnel handling animals in periods of stress, when latent carriers may reactivate the shedding of 5. Dublin. While working with animals or cleaning equipment, personal protective equipment (coveralls, washable boots, gloves, masks, and goggles) should be used. In addition, personnel should remove personal protective equipment before leaving the farm, and boots should be cleaned and disinfected.

[0096] Recent research has evaluated the possibility to use gene therapy or gene editing to inhibit virulence gene expression in Salmonella spp. The use of certain lactic acid bacteria (LAB) combinations and their degradation products has been associated with the downregulation of virulence genes in different pathogens associated with neonatal calf diarrhea, including S. Dublin. Specifically for S. Dublin, a combination of 61 LAB strains was able to downregulate the expression of virulence factor fliC, which was assessed with RT-qPCR. Additionally, CRISPR / Cas9 has been investigated to delete the plasmid-based SpvB gene by using a modified pCas9 plasmid in pathogenic strains of .S'. Gallinarum. The results are promising, as the manipulated strain did not induce clinical disease or gross pathological lesions in broiler chickens 36 days after the challenge.

[0097] Whole genome sequencing (WGS) can be used to compare the whole genome of pathogens for the surveillance and control of diseases. WGS has been used to characterize the proximity in S. Dublin clades and differentiate the AMR and MDR genes between different regions and continents. In addition, it has been used to determine the clonal relationship of 5. Dublin strains in cattle and food animal products, with the potential to track zoonotic outbreaks. Furthermore, it has been used to study the proximity between cattle and human strains of S. Dublin, their virulence, and AMR genes.

[0098] The described compositions and methods will be better understood in view of the following numbered paragraphs. 1. A method of reducing or preventing intrauterine transmission of Salmonella Dublin including a first administration, and optionally second or more administrations, to a pregnant cow of an effective amount of an immunogenic composition to induce an immune response against S. Dublin in the cow.

[0099] 2. The method of paragraph 1, wherein the cow is infected with S. Dublin.

[0100] 3. The method of paragraphs 1 or 2, wherein the subject is a latent carrier of S. Dublin.

[0101] 4. The method of any one of paragraphs 1-3, including detecting S. Dublin bacteria and / or antibodies thereto in the cow.

[0102] 5. The method of paragraph 1, wherein the cow is not infected with S. Dublin.

[0103] 6. The method of any one of paragraphs 1-5, wherein first administration is made after or coincident with confirmation of pregnancy.

[0104] 7. The method of any one of paragraphs 1-6, wherein the first administration is made in the range of 120 days or 250 days, inclusive, post-conception, or any integer subrange or specific day therebetween

[0105] 8. The method of any one of paragraphs 1-7, wherein the first administration is in the range of 120 days or 180 days, inclusive, post-conception, or any integer subrange or specific day therebetween

[0106] 9. The method of any one of paragraphs 1-7, wherein the cow is within 5-10 days of dry-off.

[0107] 10. The method of any one of paragraphs 1-7 or 9, wherein the cow is 200-250 days, optionally 215-226 days, post-conception, or any integer subrange or specific day therebetween.

[0108] 11 . The method of any one of paragraphs 1-8, including a second administration.

[0109] 12. The method of paragraph 11, wherein the second administration is 10-31, optionally 12-28, optionally 12, 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the first administration.

[0110] 13. The method of any one of paragraphs 1-12, wherein the second administration is between 155 and 287 days (i.e., 5-9 months), or between 155-218 days (i.e., 5-7 months) post-conception or any integer subrange or specific day therebetween.

[0111] 14. The method of any one of paragraphs 1-13, wherein the first administration, and optionally second or more administrations, are administered to the cow at a time(s) that reduces intrauterine transmission of S. Dublin infection from the cow to one or more of her calves compared to unvaccinated control cows and / or cows administered according to a different administration regimen.

[0112] 15. The method of any one of paragraphs 1-14, wherein the subject is administered the composition subcutaneously. 16. The method of any one of paragraphs 1-15, wherein the immunogenic composition includes an antigen derived from 5. Dublin.

[0113] 17. The method of any one of paragraphs 1-16, wherein the immunogenic composition includes an .S'. Dublin bacteria or lysate or bactrin formed therefrom.

[0114] 18. The method of paragraph 17, wherein the S. Dublin bacteria is attenuated, inactivated, and / or avirulent.

[0115] 19. The method of any one of paragraphs 1-18, wherein the immunogenic composition includes or is EnterVene®-d; Boehringer Ingelheim Animal Health.

[0116] 20. The method of any one of paragraphs 1-19, further including one or more additional steps or means of reducing or preventing S. Dublin infection in the subject.

[0117] 21. The method of any one of paragraphs 1-20, where the subject is part of a herd, and the method includes parallel treatment or prevention of other subjects in the herd.

[0118] The described compositions and methods will be better understood in view of the following experimental examples.

[0119] Examples

[0120] This randomized clinical trial was composed of 2 phases (Figure 1). The screening phase involved the identification of S. Dublin latent carriers, whereas the trial phase included the treatment allocation and sample collection.

[0121] Sample Size Determination

[0122] The sample size was determined using JMP Pro v. 17 (SAS Institute). The goal was to determine if there was a difference in the proportion of latent carrier cows shedding S. Dublin at calving between vaccinated and control cows with a power of 80%, a precision of 5%, a reduction in the proportion of shedders of at least 17% based on the findings of S. Dublin shedders in a vaccination study that used an attenuated S. Dublin bacterium expressing E. coli O157:H7 outer membrane protein (Khare et al., 2010), given the lack of S. Dublin vaccine-specific data. This resulted in a required sample size of 160 latent-carrier animals (80 per treatment; 20 cows / farm / treatment) when considering clustering in 4 different farms and a 5% attrition.

[0123] Screening Phase

[0124] A convenience sample of 4 Michigan dairy farms were used. Farm inclusion criteria included (1) a history of S. Dublin infection in the last 12 months via isolation of the bacteria from a clinical case, (2) a farm size of 1,000 milking cows or greater, (3) no current vaccination against Salmonella in cows or calves, (4) enrollment in regular Dairy Herd Improvement (DHI) testing, (5) access to cows on farm during the dry period, and (6) willingness to participate in the study. The reported average prevalence of S. Dublin latent carrier within dairy herds with a S. Dublin history was approximately 15% (Nielsen el al., 2004). Thus, in order to identify the 160 latent carriers needed, a random sample of 250 to 280 cows stratified by lactation group (1st, 2nd, or 3rdand greater) and proximity in expected calving date were screened for S. Dublin carrier status through milk ELISA at each of the 4 enrolled farms resulting in a total of 1,096 cows. .S'. Dublin, unlike other Salmonella spp., is rarely cultured from feces except in sick animals. Thus, other samples need to be examined to determine latent carrier status. A common approach to determine latent carrier status is to conduct serial testing of animals, as carriers sustain elevated antibodies against S. Dublin for long periods of time (Spier et al., 1990, Nielsen and Dohoo, 2011, Nielsen and Nielsen, 2012). For this, 3 milk samples from each cow were collected at 2-month intervals (63 to 97, 125 to 155, and 185 to 215 d pregnant) as part of the regular DHI testing, stored at -20 °C, and shipped overnight on dry ice to Cornell University Animal Health Diagnostic Center (Ithaca, NY) for 5. Dublin antibody ELISA testing (PrioCHECK™ S. Dublin Ab Strip Kit, Applied Biosystems). Following the ELISA’s manufacturer instructions, a sample was classified as positive with a percent positivity > 35%. Samples with a negative percent positivity were assigned a value of 0. Cows testing positive in all 3 tests were classified as latent carriers (Nielsen and Dohoo, 2011, Nielsen and Nielsen, 2012).

[0125] Trial Phase

[0126] The aim was to randomly select 40 latent carrier cows at each of the enrolled farms for inclusion in the vaccination trial. However, one herd was removed from the study due to the low number of latent-carrier animals identified (n=3), and all the latent carrier cows identified in 2 of the other farms were enrolled to minimize the loss of statistical power due to lower than anticipated sample size. A total of 148 (46 first, 52 second, and 50 third or greater lactation) latent carriers were included in the trial.

[0127] Animals in each herd were allocated to the vaccine or control group using randomization software (graphpad.com / quickcalcs / randomizel / ). Vaccine cows received 2 mL of a commercial live culture S. Dublin vaccine (ENTER VENE®-d; Boehringer Ingelheim Animal Health) subcutaneously (SC) within 7 d of dry-off (215 to 226 actual days pregnant) and a booster 14 d after the primary vaccination per label. Control cows received 2 mL of saline SC at the same times. Treatments were administered by research staff following the BEEF QUALITY ASSURANCE PROGRAM™ guidelines (.bqa.org / Media / BQA / Docs / nationalmanual.pdf). Farm and laboratory personnel were blinded to treatment allocation. Farm staff were trained to monitor and report adverse reactions in all enrolled cows for 4 d following the administration of treatments. All animals enrolled in the study were managed according to each farm’s protocols before, during, and after parturition. Enrolled cows were maintained in the pens with their herd mates. At calving, feces and colostrum from the enrolled cows were collected by trained farm staff into sterile containers and kept at -20 °C pending analysis. A 10 mL blood sample from the calf was also collected prior to administration of colostrum via jugular venipuncture using vacuum tubes without additives (BD vacutainer) by trained farm staff or research staff. Blood tubes were allowed to clot for 30 min, centrifuged at 1,500 g for 15 min, and the serum was harvested, aliquoted, and stored at -20 °C pending analyses.

[0128] Sample Analysis

[0129] For the qualitative identification of S. Dublin shedding, feces (1 g) and colostrum (1 mL) from latent carrier cows were cultured using a pre-enrichment method to qualitatively identify S. Dublin shedding. For this, samples were diluted 1:10 on tetrathionate broth (Beckton Dickinson) and incubated overnight (16-24 h) at 35 ± 2 °C in an aerobic atmosphere. Next, the tetrathionate broth culture was sub-cultured on brilliant green agar containing novobiocin (Hardy Diagnostics) and xylose-lysine-tergitol (ThermoFisher) agar plates. The plates were incubated overnight at 35 ± 2°C in an aerobic atmosphere. Subsequently, plates were examined to identify colonies of Salmonella spp. Isolates were confirmed employing a MicroflexLT MALDLTOF mass spectrometer (Broker Dal tonics). Whole genome sequencing based serovar prediction was performed on the isolated Salmonella sp. at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, Michigan). In brief, DNA extracted from Salmonella were quantified and library prepared using DNA Prep kit (Illumina San Diego, CA). Short-read sequencing was performed on the NextSeq 1000 Pl cartridge (Illumina San Diego, CA). The raw FASTQ files were used for serovar analysis using SeqSero 1.2 (cge.food.dtu.dk / services / SeqSero / ) (Zhang et al., 2015).

[0130] A qPCR assay was utilized to quantify the shedding of bacteria by latent carriers. The qPCR assay was made following the S'. Dublin-specific test developed by Persson et al. (2012) that targets the VagC gene. For this, DNA was extracted from the fecal and colostrum samples using a commercial kit (QIAamp Fast DNA stool mini kit; Qiagen) following the manufacturer- recommended protocol. The extracted DNA was diluted at 50 pL. A total reaction mix of 20 pL was used, composed of 10 pL of TaqMan Universal PCR Master Mix (ThermoFisher), 0.4 pL (25 pM) of each pair of primers specific for the vagC gene (Forward-GGGTGAGCGAGCTGGAAA (SEQ ID NO:1); Reverse-CGCCATAAAGTCCGGGTCA (SEQ ID NO:2)), 0.2 pL (25 pM) of probe (FAM-TTTTTCGAGCTGCGCGAACGAGC-BHQ1 (SEQ ID NO:3)), 4 pL of nuclease-free water, and 5 pL of the template DNA. DNA from an isolate of S. Dublin ATCC 15480 (American Type Culture Collection) was employed as a positive control and 5 pL of nuclease-free water were used a negative control instead of template DNA. The qPCR was run on an ABI 7500 Fast real-time PCR instrument (Thermo Fisher Scientific Inc.). The construction and standardization of the standard curves for S. Dublin genomic copy number determination was based on a 10-fold serial dilution series of the 5. Dublin ATCC 15480 isolate, ranging from 1 x 101to 1 x 10'3ng. The copy number was calculated using the following equation (Lee et al., 2006):

[0131] 6.02 x 1023 x DNA amount (g) DNA copies = - V moi / -

[0132] DNA length (dp) x 660 (g / mol / dp)

[0133] The sensitivity and specificity validation of this qPCR assay did not include several Salmonella serovars commonly isolated from cattle in the US that also express this gene. Thus, the primer annealing specificity was analyzed in silico considering the shotgun genome sequences of Salmonella Dublin (GenBank ID: JF267653.1) and other commonly isolated Salmonella vagC reported in the National Center for Biotechnology Information (NCBI) using the Unipro UGENE software Vo. 5.50 (NCBI). vagC sequences included serovars Kentucky (GenBank ID: CALNWF000000000.1), Newport (GenBank ID: AHUG01000037.1), Typhimurium (GenBank ID: CP088137.1), Braenderup (GenBank ID: UFRJ01000003.1, and Bovismorfican (GenBank ID: UGVQ01000001.1). No overlap was identified between the primers and the other serovars, confirming the specificity of the primers for S. Dublin detection.

[0134] To identify intrauterine disease transmission, pre-colostral serum samples were shipped overnight in dry ice to the Cornell University Animal Health Diagnostic Center (Ithaca, NY) for testing using a commercial S. Dublin antibody ELISA (Thermofisher). Samples were defined as positive with a percent positivity > 35%. Samples with a negative percent positivity were assigned a value of 0. Intrauterine transmission was defined when calves had a positive result on S. Dublin antibody ELISA at birth.

[0135] Statistical Analyses

[0136] All statistical analyses were completed using JMP Pro v. 17 (SAS Institute). Logistic regression models were used to analyze dichotomous data (qualitative shedding and intrauterine transmission). Linear mixed models were used to compare continuous data (quantitative assessment of bacterial shedding). Models included the fixed effect of treatment (vaccine vs. control) and the random effect of farm. The non-parametric Mann- Whitney U test compared the ELISA % positivity between calves bom to vaccinated and control dams. Statistical significance was declared at P < 0.05.

[0137] Results

[0138] Out of 1,096 cows initially enrolled for screening, samples were obtained from all 3 milk tests for 1 ,084 cows (Figure 1 ). From these, 158 (14.6%) cows were identified as latent carriers. The ELISA results of the screening phase for the cows identified as carriers are summarized in Table 1. The latent carrier within-herd prevalence varied greatly among the 4 study farms from 1.0 to 23.6% (Figure 2) despite all farms having a recent history of .S'. Dublin infection. This variation underscores the complex epidemiology of S. Dublin infection dynamics in dairy herds. The overall prevalence of latent carriers as well as the variation within herd prevalence was consistent with previous reports (Nielsen et al., 2004).

[0139] Adverse reactions to the treatments were minimal, including only the development of a 3 to 5 cm diameter swelling at the injection site (n=10; vaccine=8; control=2) that disappeared without intervention in 4 to 10 d. A total of 127 (vaccine=62, control=64) fecal, 126 (vaccine=64, control=63) colostrum, and 118 (vaccine=60, control=58) serum samples were collected and analyzed (Figure 1). Loss-to-follow-up reasons included death in the dry period (n=l; vaccine=0, control=l), dystocia (n=4; vaccine=l, control= 3), stillbirth (n=6; vaccine=3, control=3), misidentification of animals (n=10; vaccine=6 and control=4), and serum samples not collected prior to colostrum ingestion (n=9; vaccine=4 and control=5). Chi-squared analyses revealed no differences between treatment groups were found for any of the loss-to-follow-up reasons (P > 0.62). In line with a previous study (Smith et al., 2015), the commercial vaccine was found safe to use in pregnant cattle around the time of dry-off based on the lack of adverse reactions observed. Apart from the loss-to-follow-up reasons described above, all latent carriers did not develop any sign of disease that could be detected by farm staff or the rumination sensors of the farms.

[0140] A total of 12 (vaccine=2; control=10) out of 118 (10.2%) calves tested positive for .S. Dublin antibodies at birth (Table 1). Vaccination decreased the likelihood of calves being born with S. Dublin antibodies (Relative Risk [95%CI]) = 0.19 [0.04 - 0.84]; P = 0.0095). The concentration of S. Dublin antibodies, evaluated by ELISA % positivity, in calves born to vaccinated carriers was also lower (P = 0.02) than in calves bom to control carriers (Figure 3).

[0141] Intrauterine transmission of Salmonella spp. has been documented in the literature (Richardson, 1973; Hanson et al., 2016). Hanson et al. (2016) found 50% vertical transmission in infected cattle with various Salmonella enterica serovars when euthanizing calves at birth and culturing various tissues. However, the Dublin serovar was not reportedly found among the cultured and serotyped Salmonella isolates in the referenced study. The rate of .S'. Dublin vertical transmission in the control group of this study (17.2%) was lower than this previous report, but contrary to the Hanson et al. (2016) study that looked at all culturable Salmonella spp., this study focused only on the Dublin serovar. Hanson et al. (2016) also reported that 47.3% of the dams had Sa / nione / Zn-positive fecal cultures at the calving time. This prevalence of fecal shedding was almost 3 times greater than in this study (Table 3), indicating that the rate of active infections in the study might have been higher than those reported here. Pre-colostral antibody testing was used to avoid euthanizing the calves at birth. The bovine fetus starts producing antibodies in the second trimester of pregnancy (Ellis et al., 1978) and, by the third trimester, is able to produce IgG against most antigens (Banks and McGuire, 1989). Nevertheless, it would be possible for in utero infection to occur close to calving, thereby not allowing the fetus sufficient time to mount an immune response that results in measurable antibodies at birth. It is believed that this is the first report documenting the prevalence of intrauterine S. Dublin transmission from latent carrier cows, limiting the ability to compare with previous studies. The proportion of calves bom with 5. Dublin antibodies in the control group further highlights the bearing of the intrauterine transmission route on the persistence of the infection in herds and the importance of controlling .S'. Dublin latent carrier animals in infected herds. This approach, however, only assessed the fetus’ immune response and indicates intrauterine exposure. Further testing in the calves, such as pre-colostral blood or fecal cultures, would have been required to demonstrate active infection.

[0142] Table 1: Descriptive results of the ELISA % positivity in each sampling of the screening phase among the 158 Salmonella Dublin latent carriers identified.

[0143] It was additionally found that latent carriers vaccinated at dry-off with a live culture 5. Dublin commercial vaccine were 5 times less likely to give birth to a seropositive infected calf (Table 2). Because S. Dublin-infected animals carry the bacteria in their lymph nodes (Hall et al., 1978, Steinbach et al., 1996), is possible that the activation of the immune system against .S'. Dublin by immunization limited the spread of the bacteria from the lymph nodes to other tissues, resulting in a lower vertical transmission of the infection. This strategy could be used in combination with other management practices to limit the transmission of S. Dublin in cattle herds. Table 2: Contingency table of results of antibody ELISA results in pre-colostral serum samples of calves. Results are expressed as the number (%) of samples in each category.

[0144] ELISA Negative ELISA Positive Total

[0145] Control 48 (40.7%) 10 (8.5%) 58 (49.2%)

[0146] Vaccine 58 (49.1%) 2 (1.7%) 60 (50.8%)

[0147] Total 106 (89.8%) 12 (10.2%) 118 (100%)

[0148] ELISA positive samples had a percent positivity > 35%.

[0149] In feces, only 21 of the 127 samples (in 2 of the 3 farms) were positive for Salmonella spp. on culture (Table 3).

[0150] Table 3: Results of bacterial shedding analyses.

[0151] Feces Colostrum

[0152] Salmonella S. Dublin Salmonella S. Dublin culture qPCR culture qPCR

[0153] Positive 21 (16.5%) 0 (0%) 2 (1.6%) 0 (0%)

[0154] Negative 106 (83.5%) 127 (100%) 124 (98.4%) 126

[0155] (100%)

[0156] TOTAL 127 127 126 126

[0157] Results are expressed as number (%) of positive / negative samples in each test.

[0158] However, none of the isolates was S. Dublin (Table 4). Similarly, Salmonella spp. were only identified in 2 colostrum samples (in the same 2 farms). However, serotyping excluded both as not Dublin (Table 4), as did the qPCR results where none of the feces or colostrum samples were positive. Given the absence of S. Dublin bacterial identification in the latent carriers enrolled in this study, a comparison of bacterial shedding between vaccinates and controls was not undertaken.

[0159] Table 4: Whole genome sequencing based serovar prediction of all Salmonella isolates.

[0160] Salmonella Somatic (O) antigen Sample serovar formula1Feces (n=21) Colostrum (n=2)

[0161] S. Montevideo 6, 7, 14,

[0054] Total = 15 (71.4%)

[0162] Vaccine = 7 (33.3%) -

[0163] Control = 8 (38.1%)

[0164] S. Muenster 3, 10, 15, 15,34 Total = 5 (23.8%)

[0165] Vaccine = 3 (14.3%) -

[0166] Control = 2 (9.5%)

[0167] S. Cerro 6,14,18 Total = 1 (14.3%) Total = 2 (100%)

[0168] Vaccine = 0 (0.0%) Vaccine - 1 (50%)

[0169] Control = 1 (4.8%) Control = 2 (50%)

[0170] Results are expressed as number (%) of total isolates in each sample type. Results are expressed as number (%) of isolates. Given that all the cows enrolled in the trial phase were identified as latent carriers, it was believed that most of the cows in the control group would shed the bacteria at calving. The recovery of S. Dublin in feces of apparently healthy cows is usually low. However, it was considered that most of the cows in the control group would shed the bacteria at calving because only latent carriers were enrolled in the trial phase, and the stress associated with parturition increases the probability of shedding the bacteria in feces (Nielsen, 2013a). These results may reflect weaknesses in identification of latent carriers based on repeated milk antibody ELISA testing, the sensitivity of the methods available to detect .S'. Dublin via bacteriology, and / or the relevance of bacterial shedding from latent carriers at the time of calving on disease transmission. Nielsen (2013b) also reported a low prevalence of fecal shedders among cattle with persistently high S. Dublin antibodies, although these animals were not sampled around a stressful event. Repeated sampling could have increased the ability to identify shedders by fecal culture. However, repeated culture before or after calving was logistically not viable under the conditions of this study and, to a certain degree, shedding at different times than calving would have limited impact on disease transmission to the newborn in systems where dams and calves are separated shortly after birth (discussed below).

[0171] Identification of latent carrier cows is based on serology because the intermittent shedding of S. Dublin by these animals makes bacteria culture and identification methods unreliable (Smith et al., 1992). However, the classification of animals based on antibody concentration results varies in the literature. Most researchers recommend serial testing of animals to differentiate between recent exposure and carrier status because the antibody response of transiently infected animals may take months to decrease to undetectable levels using ELISA (Smith et al., 1989; Spier et al., 1990). However, the number and frequency of tests required to define an animal as a carrier through serial testing remains controversial, with some authors indicating that 2 positive ELISA tests taken 60 d apart is sufficient to confidently classify S. Dublin latent-carriers (Spier et al., 1990). In this study, however, a more conservative approach was taken, and 3 positive tests 2 months apart were required to define latent carrier status, in line with other recommendations (Smith et al., 1992; Nielsen et al., 2004).

[0172] The commercial ELISA assay used targets antibodies directed against the Salmonella spp LPS O-antigens 1, 9, and 12. Thus, there is some possibility for cross-reaction among Salmonella serovars (Konrad et al., 1994), which could have impacted the results and the classification of latent carriers. However, lack of cross-reactivity has also been reported (Agren et al., 2016), and none of the isolates identified in the study expressed any of the antigens included in the ELISA assay (Table 4). Thus, it is believed that ELISA cross-reactivity might have had a limited impact in the results. Furthermore, the serial testing approach used is intended to minimize the impact of potential ELISA cross-reactivity. Also, a more conservative approach was taken by using the ELISA assay’s manufacturer recommended cut-off percent positivity of 35% despite some evidence in the literature that a 20% cut-off could be sufficient to detect latent carriers (Nyman et al., 2013).

[0173] Common Salmonella culture methods were utilized for the isolation of S. Dublin in feces and colostrum that require pre-enrichment and subsequent selective media culture to increase bacterial detection (ISO, 2017). Nevertheless, only 16.5% and 1.6% of fecal and colostrum samples, respectively, had a positive Salmonella culture (Table 3) despite all enrolled cows having sustained Salmonella antibody titers prior to calving. Individual animal fecal culture for S. Dublin is thought to have a sensitivity of 16-20% (Nielsen, 2013). This relatively low sensitivity coupled with the sporadic nature of shedding, and the fact that latent carrier cattle shed lower numbers of organisms than clinically ill or acutely infected animals, along with only sampling cows once, might explain the low detection observed in this study via culture. Furthermore, cattle can be coinfected with more than one Salmonella serovar (Hanson et al., 2016). In those instances, the intraspecies gut competition may increase the longitudinal shedding and colony-forming unit counts from the non-host adapted serotypes over those host-adapted like Dublin (Kirchner et al., 2012). However, based on the one-time sampling, the lack of S. Dublin detection on direct fecal / colostrum PCR indicates that S. Dublin shedding was non-existent or below the PCR detection limit. qPCR analyses were conducted to (1) identify the bacteria based on detection of genetic material, and (2) estimate the number of bacteria being shed as the required pre-enrichment stage in the culture protocol precludes a direct quantification of colony forming units. Molecular biology techniques have been recommended to be included to complement bacteria culture in Salmonella spp. field studies to increase diagnostic ability (Jensen et al., 2013). Thus, PCR specific for the Dublin serovar (Persson et al., 2012) were used. Despite these efforts, S. Dublin was not identified in fecal or colostrum samples. Overall, the limitations of the bacterial identification techniques available and the shedding pattern of the bacteria, and only sampling cows at one point, precluded evaluation of the extent to which dry cow vaccination could have decreased .S'. Dublin shedding.

[0174] Ultimately, the results also question the role of S. Dublin latent carriers in transmitting the disease to newborn calves in the maternity area. Latent carriers are believed to play a role in the transmission of the disease to newborn calves via excretion of the bacteria in feces and colostrum (Nielsen, 2013; Velasquez-Munoz et al., 2024). However, S. Dublin was not isolated in any of the fecal or colostrum samples collected at the time of calving. Although it is possible that shedding occurred later than when samples were collected, 56.2% of US dairy farms separate calves and cows within 6 h of birth, and 24.5% of farms with 500 or more cattle within 1 h (NAHMS, 2014). Thus, delayed bacterial shedding relative to calving would have a limited impact on the transmission of disease to newborns provided the maternity area is maintained clean between animals because calves would be removed from maternity in most farms before cows start shedding bacteria. This is consistent with a recent cross-sectional study that found that increasing the frequency of adding bedding to the maternity pen was associated with decreased odds of the farm being positive for S. Dublin (Perry et al., 2023).

[0175] Conclusions

[0176] Vaccinating of S. Dublin latent carrier cows at dry-off with a commercial live culture vaccine reduced intrauterine transmission to calves by 81% based on the presence of precolostral S. Dublin antibodies. This strategy could contribute to decreasing the transmission of S. Dublin in dairy farms.

[0177] References

[0178] Agren, E. C., S. Sternberg Lewerin, H. Wahlstrom, U. Emanuelson, and J. Frossling. 2016. Low prevalence of Salmonella in Swedish dairy herds highlight differences between serotypes. Prev. Vet. Med. 125:38-45. doi.org / 10.1016 / j.prevetmed.2015.12.015

[0179] Costa, L. F., T. A. Paixao, R. M. Tsolis, A. J. Baumler, and R. L. Santos. 2012. Salmonellosis in cattle: advantages of being an experimental model. Res Vet Sci 93(1): 1-6. doi.org / 10.1016 / j.rvsc.2012.03.002

[0180] Counter, D. E. and E. A. Gibson. 1980. Salmonella Dublin infection in self contained dairy herds in East Anglia: excretion at calving. Vet. Rec. 107(9): 191 -193. doi.org / 10.1136 / vr.l07.9.191 Cummings, K. J., L. D. Warnick, K. A. Alexander, C. J. Cripps, Y. T. Grohn, P. L.

[0181] McDonough, D. V. Nydam, and K. E. Reed. 2009. The incidence of salmonellosis among dairy herds in the northeastern United States. J. Dairy Sci. 92(8):3766-3774. doi.org / 10.3168 / jds.2009- 2093

[0182] Cummings, K. J., L. D. Warnick, M. Elton, Y. T. Grohn, P. L. McDonough, and J. D. Siler. 2010. The effect of clinical outbreaks of salmonellosis on the prevalence of fecal Salmonella shedding among dairy cattle in New York. Foodborne Pathog Dis 7(7):815-823. doi.org / 10.1089 / fpd.2009.0481

[0183] Erdman, M., K. Lantz, B. R. Morningstar-Shaw, D. A. Barker, T. A. Mackie, M. I. Munoz, E. A. Palmer, and L. K. Cox. 2012. Salmonella serotypes isolated from animals in the United States. Pages 435-441 in Proc. 116th Annual Meeting US Animal Health Association, Greensboro, NC.

[0184] FDA, Food and Drug Administration. 1994. Animal Medicinal Drug Use Clarification Act of 1994 (AMDUCA), Title 21, Code of Federal Regulations, Part 530.41. .ecfr.gov / current / title- 21 / section-530.41 Hanson, D. L., G. H. Loneragan, T. R. Brown, D. J. Nisbet, M. E. Hume, and T. S. Edrington. 2016. Evidence supporting vertical transmission of Salmonella in dairy cattle. Epidemiol Infect 144(5):962-967. doi.org / 10.1017 / S0950268815002241

[0185] Helms, M., P. Vastrup, P. Gemer-Smidt, and K. Molbak. 2003. Short and long term mortality associated with foodborne bacterial gastrointestinal infections: registry based study. BMJ 326(7385):357. doi.org / 10.1136 / bmj.326.7385.357

[0186] Holschbach, C. L. and 5. F. Peek. 2018. Salmonella in Dairy Cattle. Vet. Clin. North. Am. Food Anim. Pract. 34(1): 133- 154. doi.org / 10.1016 / j.cvfa.2017.10.005

[0187] ISO, International Organization for Standardization. 2017. Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1 : Detection of Salmonella spp. (ISO Standard No. 6579-1 :2017). .iso.org / standard / 56712.html. Accessed on: 10 / 10 / 2023

[0188] Khare, S., W. Alali, S. Zhang, D. Hunter, R. Pugh, F. C. Fang, S. J. Libby, and L. G. Adams. 2010. Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle. BMC Vet. Res. 6(1):35. doi.org / 10. 1186 / 1746-6148-6-35

[0189] Konrad, H., B. P. Smith, G. W. Dilling, and J. K. House. 1994. Production of Salmonella serogroup D (O9)-specific enzyme-linked immunosorbent assay antigen. Am. J. Vet. Res. 55(12): 1647-1651.

[0190] Lee, C., J. Kim, .S'. G. Shin, and S. Hwang. 2006. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123(3):273-280. doi.org / 10.1016 / j.jbiotec.2005.11.014

[0191] Munoz, N., M. Diaz-Osorio, J. Moreno, M. Sanchez- Jimenez, and N. Cardona-Castro. 2010. Development and evaluation of a multiplex real-time polymerase chain reaction procedure to clinically type prevalent Salmonella enterica serovars. J Mol Diagn 12(2):220-225. doi.org / 10.2353 / jmoldx.2010.090036

[0192] NAHMS, National Animal Health Monitoring Service 2014. Dairy 2014. Part 1 : Reference of Dairy Health and Management in the United States. USDA: APHIS Veterinary Services, Ft, Collins, C.O.

[0193] Nielsen, L. R., Y. H. Schukken, Y. T. Grohn, and A. K. Ersboll. 2004. Salmonella Dublin infection in dairy cattle: risk factors for becoming a carrier. Prev. Vet. Med. 65(l-2):47-62. doi.org / 10.1016 / j.prevetmed.2004.06.010

[0194] Nielsen, L. R. and I. Dohoo. 2011. Culling decisions of dairy farmers during a 3-year Salmonella control study. Prev. Vet. Med. 100(1 ):29-37. doi.org / 10.1016 / j.prevetmed.2011.03.001 Nielsen, L. R. and .S'. S. Nielsen. 2012. A structured approach to control of Salmonella Dublin in 10 Danish dairy herds based on risk scoring and test-and-manage procedures. Food Res Int 45(21:1158-1165. doi.org / 10.1016 / j.foodres.2011.02.027

[0195] Nielsen, L. R. 2013. Review of pathogenesis and diagnostic methods of immediate relevance for epidemiology and control of Salmonella Dublin in cattle. Vet Microbiol 162(1): 1-9. doi.org / 10.1016 / j.vetmic.2012.08.003

[0196] Nyman, A. K., E. C. Agren, K. Bergstrom, and H. Wahlstrom. 2013. Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk. Acta Vet. Scand. 55(1):5. doi.org / 10.1186 / 1751-0147-55-5

[0197] Paudyal, N., H. Pan, M. Elbediwi, X. Zhou, X. Peng, X. Li, W. Fang, and M. Yue. 2019. Characterization of Salmonella Dublin isolated from bovine and human hosts. BMC Microbiol 19(1):226. doi.org / 10. 1186 / S12866-019-1598-0

[0198] Perry, K. V, D. F. Kelton, S. Dufour, C. Miltenburg, S. G. U. Sedo, and D. L. Renaud. 2023. Risk factors for Salmonella Dublin on dairy farms in Ontario, Canada. J. Dairy Sci. doi.org / 10.3168 / jds.2023-23517

[0199] Persson, S., T. Jacobsen, J. E. Olsen, K. E. Olsen, and F. Hansen. 2012. A new real-time PCR method for the identification of Salmonella Dublin. J Appl Microbiol 113(3):615-621. doi.org / 10.1111 / j.1365-2672.2012.05378. x

[0200] Richardson, A. 1973. The transmission of Salmonella Dublin to calves from adult carrier cows. Vet. Rec. 92(5): 112- 115. doi.org / 10.1136 / vr.92.5.112

[0201] Sharma, P., C. Caraguel, M. Sexton, A. McWhorter, G. Underwood, K. Holden, and K. Chousalkar. 2018. Shedding of Salmonella Typhimurium in vaccinated and unvaccinated hens during early lay in field conditions: a randomised controlled trial. BMC Microbiol 18(1) :78. doi.org / 10.1186 / s 12866-018- 1201 -0

[0202] Smith, B., J. House, G. Dilling, L. Roden, and S. Spier. 1992. Identification of Salmonella Dublin carrier cattle. Pages 225-230 in Proc. Proceedings of the International Symposium Salmonella and Salmonellosis. Zoopole, Ploufragan France.

[0203] Smith, B. P., D. G. Oliver, P Singh, G. Dilling, P. A. Martin, B. P. Ram, L. S'. Jang, N. Sharkov, J. S. Orsborn, and et al. 1989. Detection of Salmonella Dublin mammary gland infection in carrier cows, using an enzyme-linked immunosorbent assay for antibody in milk or serum. Am. J. Vet. Res. 50(8): 1352- 1360.

[0204] Smith, G. W., F. Smith, S. Zuidhof, and D. M. Foster. 2015. Short communication: Characterization of the serologic response induced by vaccination of late-gestation cows with a Salmonella Dublin vaccine. J. Dairy Sci. 98(4):2529-2532. doi.org / 10.3168 / jds.2014-8972 Spier, 5. J., B. P. Smith, J. W. Tyler, J. S. Culler, G. W. Dilling, and L. Da Pfaff. 1990. Use of ELISA for detection of immunoglobulins G and M that recognize Salmonella Dublin lipopolysaccharide for prediction of carrier status in cattle. Am. J. Vet. Res. 51 (12): 1900- 1904.

[0205] Syed, K. A., T. Saluja, H. Cho, A. Hsiao, H. Shaikh, T. A. Wartel, V. Mogasale, J. Lynch, J. H. Kim, J. L. Excler, and S. Sahastrabuddhe. 2020. Review on the Recent Advances on Typhoid Vaccine Development and Challenges Ahead. Clin Infect Dis 71(Suppl 2):S141-S150. doi.org / 10. 1093 / cid / ciaa504

[0206] Valenzuela, J. R., A. K. Sethi, N. A. Aulik, and K. P. Poulsen. 2017. Antimicrobial resistance patterns of bovine Salmonella enterica isolates submitted to the Wisconsin Veterinary Diagnostic Laboratory: 2006-2015. J. Dairy Sci. 100(2):1319-1330. doi.org / 10.3168 / jds.2016-11419

[0207] Velasquez-Munoz, A., R. Castro- Vargas, F. M. Cullens-Nobis, R. Mani, and A. Abuelo. 2024. Review: Salmonella Dublin in dairy cattle. Front. Vet. Sci. 10: 1331767. doi.org / 10.3389 / fvets.2023.1331767

[0208] Zhang, S., Y. Yin, M. B. Jones, Z. Zhang, B. L. Deatherage Kaiser, B. A. Dinsmore, C. Fitzgerald, P. I. Fields, and X. Deng. 2015. Salmonella serotype determination utilizing high- throughput genome sequencing data. J. Clin. Microbiol. 53(5): 1685-1692. doi.org / 10.1128 / JCM.00323-15

[0209] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0210] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific forms of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of reducing or preventing intrauterine transmission of Salmonella enterica serotype Dublin (S. Dublin) comprising a first administration, and optionally second or more administrations, to a pregnant cow of an effective amount of an immunogenic composition to induce an immune response against 5. Dublin in the cow.

2. The method of claim 1, wherein the cow is infected with .S'. Dublin.

3. The method of claim 1, wherein the cow is a latent carrier of S. Dublin.

4. The method of claim 1, comprising detecting S. Dublin bacteria and / or antibodies thereto in the cow.

5. The method of claim 1, wherein the cow is not infected with 5. Dublin.

6. The method of claim 1 , wherein first administration is made after or coincident with confirmation of pregnancy in the cow.

7. The method of claim 1, wherein the first administration is made in the range of 120 days or 250 days, inclusive, post-conception, or any integer subrange or specific day therebetween8. The method of claim 1, wherein the first administration is in the range of 120 days or 180 days, inclusive, post-conception, or any integer subrange or specific day therebetween.

9. The method of claim 1, wherein the cow is within 5-10 days, inclusive, of dry-off.

10. The method of claim 1, wherein the cow is 200-250 days, inclusive post-conception, or any integer subrange or specific day therebetween, optionally wherein the cow is 215-226 days, inclusive, post-conception, or any integer subrange or specific day therebetween.

11. The method of claim 1 , comprising a second administration.

12. The method of claim 11, wherein the second administration is 10-31 days, inclusive, after the first administration, optionally wherein the second administration is 12-28 days, inclusive, after the first administration, further optionally wherein the second administration is 12, 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the first administration.

13. The method of claim 11, wherein the second administration is between 155 and 287 days, inclusive, post-conception or any integer subrange or specific day therebetween, or wherein the second administration is between 155-218 days, inclusive, post-conception or any integer subrange or specific day therebetween.

14. The method of claim 1, wherein the first administration, and optionally second or more administrations, are administered to the cow at a time(s) that reduces intrauterine transmission of .S'. Dublin infection from the cow to one or more of her calves compared to unvaccinated control cows and / or cows administered according to a different administration regimen.

15. The method of claim 1, wherein the cow is administered the composition subcutaneously.

16. The method of claim 1 , wherein the immunogenic composition comprises an antigen derived from S. Dublin.

17. The method of claim 1, wherein the immunogenic composition comprises an 5. Dublin bacterium, or lysate, or bactrin formed therefrom.

18. The method of claim 17, wherein the S. Dublin bacterium is attenuated, inactivated, and / or avirulent.

19. The method of claim 1, wherein the immunogenic composition comprises or is ENTER VENE®-d; Boehringer Ingelheim Animal Health.

20. The method of claim 1, further comprising one or more additional steps or means of reducing or preventing S. Dublin infection in the cow.

21. The method of claim 1, wherein the cow is part of a herd, and wherein the method comprises parallel treatment or prevention of other cows in the herd.

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