Search

Since the initial use of vaccination in the eighteenth century, our understanding of human and animal immunology has greatly advanced and a wide range of vaccine technologies and delivery systems have been developed. The COVID-19 pandemic response leveraged these innovations to enable rapid development of candidate vaccines within weeks of the viral genetic sequence being made available. The development of vaccines to tackle emerging infectious diseases is a priority for the World Health Organization and other global entities. More than 70% of emerging infectious diseases are acquired from animals, with some causing illness and death in both humans and the respective animal host. Yet the study of critical host–pathogen interactions and the underlying immune mechanisms to inform the development of vaccines for their control is traditionally done in medical and veterinary immunology ‘silos’. In this Perspective, we highlight a ‘One Health vaccinology’ approach and discuss some key areas of synergy in human and veterinary vaccinology that could be exploited to accelerate the development of effective vaccines against these shared health threats., Emerging diseases that affect humans often arise due to the crossover of infectious agents from animal reservoirs. In this Perspective, George Warimwe and colleagues discuss the concept of ‘One Health vaccinology’, an approach that aims to use key lessons from human and veterinary immunology to develop more effective vaccination strategies for emerging infectious diseases.

Several observations in the world of comparative immunology in plants, insects, fish and eventually mammals lead to the discovery of trained immunity in the early 2010's. The first demonstrations provided evidence that innate immune cells were capable of developing memory after a first encounter with some pathogens. Trained immunity in mammals was initially described in monocytes with the Bacille Calmette-Guerin vaccine (BCG) or prototypical agonists like β-glucans. This phenomenon relies on epigenetic and metabolic modifications leading to an enhanced secretion of inflammatory cytokines when the host encounters homologous or heterologous pathogens. The objective of our research was to investigate the trained immunity, well-described in mouse and human, in other species of veterinary importance. For this purpose, we adapted an in vitro model of trained innate immunity in dogs. Blood enriched monocytes were stimulated with β-glucans and we confirmed that it induced an increased production of pro-inflammatory and anti-microbial compounds in response to bacterial stimuli. These results constitute the first demonstration of trained immunity in dogs and confirm its signatures in other mammalian species, with an implication of cellular mechanisms similar to those described in mice and humans regarding cellular epigenetics and metabolic regulations.

It is well-recognised that research capability in veterinary species is restricted by a lack of immunological reagents relative to theextensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect humanhealth by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focussing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) ImmuneToolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The futureavailability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccinedesign as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

Lymphatic cannulation models are useful tools for studying the immunobiology of the lymphatic system and the immunopathology of specific tissues in diseases. Sheep cannulations have been used extensively, as models for human physiology, fetal and neonatal development, human diseases, and for studies of ruminant pathobiology. The development of new and improved cannulation techniques in recent years has meant that difficult to access sites, such as mucosal associated tissues, are now more readily available to researchers. This review highlights the new approaches to cannulation and how these, in combination with advanced omics technologies, will direct future research using the sheep model.

Significant progress has been made over the last decade in defining major histocompatibility complex (MHC) diversity at the nucleotide, allele, haplotype, diplotype, and population levels in many non-human species. Much of this progress has been driven by the increased availability and reduced costs associated with nucleotide sequencing technologies. This report provides an update on the activities of the comparative MHC nomenclature committee which is a standing committee of both the International Society for Animal Genetics (ISAG) and the International Union of Immunological Societies (IUIS) where it operates under the umbrella of the Veterinary Immunology Committee (VIC). A previous report from this committee in 2006 defined the role of the committee in providing guidance in the development of a standardized nomenclature for genes and alleles at MHC loci in non-human species. It described the establishment of the Immuno Polymorphism Database, IPD-MHC, which continues to provide public access to high quality MHC sequence data across a range of species. In this report, guidelines for the continued development of a universal MHC nomenclature framework are described, summarizing the continued development of each species section within the IPD-MHC project.

Fasciola hepatica is a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response to F. hepatica following natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed to F. hepatica infection. Cohorts of replacement dairy heifer calves (n = 42) with no prior exposure to F. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through an F. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge with F. hepatica. This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived ‘infection’. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break ‘immunological ignorance’. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.

In veterinary animal species, vaccines are the primary tool for disease prevention, a key tool for treatment of infection, and essential for helping maintain animal welfare and productivity. Traditional vaccine development by trial-and-error has achieved many successes. However, effective vaccines that provide solid cross-protective immunity with excellent safety are still needed for many diseases. The path to development of vaccines against difficult pathogens requires recognition of uniquely evolved immunological interactions of individual animal hosts and their specific pathogens. Here, general principles that currently guide veterinary immunology and vaccinology research are reviewed, with an emphasis on examples from swine. Advances in genomics and proteomics now provide the community with powerful tools for elucidation of regulatory and effector mechanisms of protective immunity that provide new opportunities for successful translation of immunological discoveries into safe and effective vaccines.

High metabolism and body temperatures of flying bats might enable them to host many viruses., Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. We hypothesize that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. On an evolutionary scale, this host–virus interaction might have resulted in the large diversity of zoonotic viruses in bats, possibly through bat viruses adapting to be more tolerant of the fever response and less virulent to their natural hosts.

The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38–0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16–2.15 g/dL) and 0.40–0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18–1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology.

A comparative study was conducted using data from naive bison ( n = 45) and cattle ( n = 46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered during midgestation. The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater ( P < 0.05) in bison than in cattle. In animals that did abort, the time between experimental challenge and abortion was shorter ( P < 0.05) for bison than for cattle. Brucella colonization of four target tissues and serologic responses on the standard tube agglutination test at the time of abortion did not differ ( P > 0.05) between cattle and bison. The results of our study suggest that naive bison and cattle have similarities and differences after experimental exposure to a virulent B. abortus strain. Although our data suggest that bison may be more susceptible to infection with Brucella , some pathogenic characteristics of brucellosis were similar between bison and cattle.

Porcine circovirus type 2 (PCV2) vaccines have become widely used since they became available in 2006. It is not uncommon for producers to use PCV2 vaccines in pigs younger than what is approved by manufacturers. The objective of this study was to determine the efficacy of a chimeric and a subunit PCV2 vaccine administered at 5 or 21 days of age. Forty-eight PCV2-naïve piglets were randomly divided into six groups of eight pigs each. Vaccination was done at day 5 or day 21, followed by triple challenge with PCV2, porcine parvovirus (PPV), and porcine reproductive and respiratory syndrome virus (PRRSV) at day 49. Vaccinated pigs seroconverted to PCV2 approximately 14 days postvaccination and had a detectable neutralizing antibody response by 21 days postvaccination regardless of age at vaccination. At day 49, the pigs vaccinated with the chimeric vaccine had significantly higher levels of neutralizing antibodies than the pigs vaccinated with the subunit vaccine. After challenge, vaccinated pigs had significantly decreased levels of PCV2 viremia and a decreased prevalence and severity of microscopic lesions compared to the positive-control group, which had severe lymphoid lesions associated with abundant PCV2 antigen, compatible with PCV-associated disease. The results of this study indicate that, under the conditions of this study, vaccination of PCV2-naïve pigs at day 5 or day 21 resulted in development of a detectable humoral immune response and provided reduction or complete protection against PCV2 viremia and PCV2-associated lesions after triple challenge with PCV2, PPV, and PRRSV.

Classical swine fever virus (CSFV) and porcine parvovirus (PPV) are highly contagious pathogens, resulting in enormous economic losses in pig industries worldwide. Because vaccines play an important role in disease control, researchers are seeking improved vaccines that could induce antiviral immune responses against CSFV and PPV at the mucosal and systemic levels simultaneously. In this study, a genetically engineered Lactobacillus strain coexpressing the CSFV-specific cytotoxic T lymphocyte (CTL) epitope 290 and the VP2 antigen of PPV was developed, and its immunopotentiating capacity as an oral vaccine in pigs was analyzed. The data demonstrated that in the absence of any adjuvant, the recombinant Lactobacillus strain can efficiently stimulate mucosal and systemic CSFV-specific CD8 + CTL responses to protect pigs against CSFV challenge. Moreover, anti-PPV-VP2 serum IgG and mucosal IgA were induced in pigs immunized orally with the recombinant Lactobacillus strain, showing a neutralizing effect on PPV infection. The results suggest that the recombinant Lactobacillus microecological agent may be a valuable component of a strategy for development of a vaccine against CSFV and PPV.

Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohen’s kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals. Brucellosis, a contagious disease primarily affecting animals, is caused by members of the genus Brucella, namely, Brucella abortus (cattle, bison, and buffalo), B. melitensis (goats and sheep), B. suis (swine), B. canis (dogs), B. ovis (sheep), and B. neotomae (rodents). Bovine brucellosis is caused by biovars of B. abortus. The disease is asymptomatic in animals that have not conceived. Following infection with B. abortus, pregnant adult females develop placentitis, resulting in abortion between the fifth and ninth months of pregnancy and infertility. Adult male cattle may develop orchitis, leading to infertility. Brucellosis has zoonotic potential in terms of its transmissibility to human beings attending infected livestock, showing symptoms in the form of low-grade undulant fever, night sweats, early fatigue, joint pain leading to spondylitis, and orchitis, and it is commonly known as “undulant fever,” “Mediterranean fever,” or “Malta fever” (7). The disease remains a public health problem and results in severe economic losses in

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 10 5 CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia. The bighorn sheep (BHS; Ovis canadensis) population in North America has declined drastically during the last century due to a combination of factors, including loss of habitat, competition for forage with domestic livestock, predation, and disease. Pneumonia is the primary disease that causes significant mortality in BHS (2, 26). Although Mannheimia haemolytica, Bibersteinia trehalosi, and Pasteurella multocida have been isolated from several pneumonia outbreaks, only M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions (4, 7, 9, 22). Virulence factors of M. haemolytica include capsule, outer membrane proteins, neuraminidase, lipopolysaccharide, and a potent exotoxin called leukotoxin (Lkt), which is cytolytic to all subsets of ruminant leukocytes (3, 14). Based on the fact that Lkt deletion mutants cause no mortality in BHS (4) and lower mortality and mild lung lesions in cattle (12, 19, 24), Lkt has been accepted as the major virulence factor of this organism. Although M. haemolytica causes pneumonia in all ruminants

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.

Glanders, caused by the Gram-negative, nonmotile bacteriumBurkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance andB. malleiis listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinantBurkholderiaintracellular motility A (rBimA) protein inEscherichia colifor the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

Commensal microbes in the intestine are in constant interaction with host cells and play a role in shaping the immune system.Lactobacillus acidophilus,Lactobacillus reuteri, andLactobacillus salivariusare members of the chicken intestinal microbiota and have been shown to induce different cytokine profiles in mononuclear cellsin vitro. The objective of the present study was to examine the effects of these bacteria individually or in combination on the induction of antibody- and cell-mediated immune responsesin vivo. The birds received lactobacilli weekly via oral gavage starting on day of hatch and subsequently, at 14 and 21 days, were immunized with sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), Newcastle disease virus vaccine, and infectious bursal disease virus vaccine. Antibody responses in serum were measured weekly for 4 weeks beginning on the day of primary immunization. The cell-mediated immune response was evaluated at 21 days postimmunization by measurement of gamma interferon (IFN-γ) production in splenocytes stimulated with inactivated vaccine antigens.L. salivarius-treated birds had significantly more serum antibody to SRBC and KLH than birds that were not treated with probiotics.L. salivarius-treated birds also had decreased cell-mediated immune responses to recall antigen stimulation.L. reuteritreatment did not significantly affect the systemic immune response, whileL. acidophilustreatment increased the antibody response to KLH. These results indicate that systemic antibody- and cell-mediated immune responses can be modulated by oral treatment with lactobacilli but that these bacteria may vary in their ability to modulate the immune response.

Investigation of antigenic determinants of the microaerophilic obligate intracellular bacteriumLawsonia intracellularisusing a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infectedin vitrocell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled fromL. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ∼72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotatedL. intracellularisgenome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsoniaautotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies onin vitroculture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.

The objective of the present study was to determine the effect of an inactivated porcine circovirus type 2 (PCV2) vaccine on PCV2b virus shedding in the semen of experimentally infected boars by measuring the immunological response and the PCV2b DNA load in blood and semen. Twelve boars were randomly divided into three groups. The boars in group 1 (n= 4) were immunized with an inactivated PCV2 vaccine and were challenged with PCV2b. The boars in group 2 (n= 4) were only challenged with PCV2b. The boars in group 3 (n= 4) served as negative controls. The number of PCV2 genome copies of PCV2 in the serum and semen were significantly lower in vaccinated challenged boars than in nonvaccinated challenged boars at 7, 10, 14, 21, 32, 35, 42, 49, and 60 days postinoculation. The number of PCV2b genomes in the semen correlated with the number of PCV2b genomes in the blood in both vaccinated challenged (R= 0.714) and nonvaccinated challenged (R= 0.861) boars. The results of the present study demonstrate that the inactivated PCV2 vaccine significantly decreases the amount of PCV2b DNA shedding in semen from vaccinated boars after experimental infection with PCV2b.

Mannheimia haemolytica consistently causes fatal bronchopneumonia in bighorn sheep (BHS; Ovis canadensis) under natural and experimental conditions. Leukotoxin is the primary virulence factor of this organism. BHS are more susceptible to developing fatal pneumonia than the related species Ovis aries (domestic sheep [DS]). In BHS herds affected by pneumonia, lamb recruitment is severely impaired for years subsequent to an outbreak. We hypothesized that a lack of maternally derived antibodies (Abs) against M. haemolytica provides an immunologic basis for enhanced susceptibility of BH lambs to population-limiting pneumonia. Therefore, the objective of this study was to determine the titers of Abs directed against M. haemolytica in the sera of BH and domestic lambs at birth through 12 weeks of age. Results revealed that BH lambs had approximately 18-fold lower titers of Ab against surface antigens of M. haemolytica and approximately 20-fold lower titers of leukotoxin-neutralizing Abs than domestic lambs. The titers of leukotoxin-neutralizing Abs in the serum and colostrum samples of BH ewes were approximately 157- and 50-fold lower than those for domestic ewes, respectively. Comparatively, the higher titers of parainfluenza 3 virus-neutralizing Abs in the BH lambs ruled out the possibility that these BHS had an impaired ability to passively transfer Abs to their lambs. These results suggest that lower levels of leukotoxin-neutralizing Abs in the sera of BH ewes, and resultant low Ab titers in their lambs, may be a critical factor in the poor lamb recruitment in herds affected by pneumonia. The bighorn sheep (BHS; Ovis canadensis) population of North America has declined from an estimated 2 million animals at the beginning of the 19th century to less than 70,000 at this time (2, 32). Factors contributing to this population decline include predation, loss of habitat, competition for forage with livestock, and respiratory disease. Outbreaks of bronchopneumonia in previously healthy populations of BHS often result in high death rates among all age groups initially, followed by years of impaired recruitment due to pneumonia in lambs (5, 21, 24, 28). During these outbreaks, members of the genera Mannheimia, Bibersteinia, and Pasteurella, including Mannheimia (Pasteurella) haemolytica, Bibersteinia (Pasteurella) trehalosi, and Pasteurella multocida, have commonly been isolated from pneumonic lungs (16, 26). Of these, M. haemolytica has consistently been shown to cause fatal bronchopneumonia in BHS under experimental conditions (9, 12, 20). While M. haemolytica can cause pneumonia in multiple ruminant species, including cattle and domestic sheep (DS), BHS are particularly susceptible to this disease. A number of studies have shown that BHS demonstrate greater susceptibility to M. haemolytica, and exhibit more severe pathology when infected, than DS (9, 11, 12, 24).

Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination.

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

The effects of the recent vaccinations against bluetongue virus serotype 1 (BTV-1) and BTV-8 in Europe on the reliability of enzyme-linked immunosorbent assays (ELISAs) currently used for diagnosis of small-ruminant lentivirus (SRLV) infection were examined. Primary vaccination against BTV-8 in goats induced an increase in reactivity that did not exceed 3 months in a whole-virus indirect ELISA and a competitive ELISA based on the gp135 glycoprotein. Subsequent BTV-1/8 vaccination extended the time scale of false-positive reactivity for up to 6 months. These results are of relevance for SRLV-monitoring programs.

Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat ( Felis catus ) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.

Since first emerging in the North American canine population in 2004, canine influenza virus (CIV) subtype H3N8 has shown horizontal transmission among dogs, with a high level of adaptation to this species. The severity of disease is variable, and coinfection by other respiratory pathogens is an important factor in the degree of morbidity and mortality. The first influenza vaccine for dogs, an inactivated vaccine containing CIV subtype H3N8, was conditionally approved by the U.S. Department of Agriculture (USDA) for licensure in May 2009 and fully licensed in June 2010. This study evaluates the efficacy of this vaccine to reduce the severity of illness in dogs cochallenged with virulent CIV andStreptococcus equisubsp.zooepidemicus.

Chronic infection of cattle withLeptospira borgpeterseniiserovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4+, CD8+, and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigenex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly toL. borgpeterseniiserovar Hardjo infection may be an important step toward developing protective immunity.

Rickettsia japonica pathogenesis and reservoir potential in dogs were evaluated by both experimental inoculation and epidemiologic survey. In the experimental inoculation study, dogs 1 and 2 were pretreated with an immunosuppressive dose of cyclosporine 14 days before inoculation and became ill after exposure to R. japonica. Dogs exhibited clinical signs, including fever, anorexia, depression, and decreased water consumption, between 36 and 96 h after inoculation, but these signs disappeared spontaneously by 5 days after inoculation. Dogs 3 and 4 were not pretreated with cyclosporine, and no clinical signs were detected in them throughout the 14-day observation period. The control dog was clinically normal and had a normal rectal temperature throughout the study period. We attempted to detect rickettsial DNA from peripheral blood and aspiration samples from kidney and spleen by nested PCR, but all samples examined were negative. The control dog lacked detectable titers to R. japonica antigen on day 14, while positive antibodies to R. japonica were detected in all four experimentally infected dogs, with titers of 1:160 to 1:80. In the epidemiologic survey, 24 (1.8%) of the 1,363 dogs examined throughout Japan had antibodies against R. japonica , with titers of 1:40 or more. However, we observed neither clinical signs at the time of sample collection nor nested PCR results indicative of rickettsial infection in these dogs. In conclusion, dogs in Japan can be exposed to R. japonica , and infected dogs with immunosuppressive conditions can temporarily develop clinical symptoms, including fever, anorexia, depression, and decreased water consumption.

The objectives of this study were to compare relative vaccine-specific serum immunoglobulin concentrations, vaccine-specific lymphoproliferative responses, and cytokine profiles of proliferating lymphocytes between 3-day-old foals, 3-month-old foals, and adult horses after vaccination with a killed adjuvanted vaccine. Horses were vaccinated intramuscularly twice at 3-week intervals with a vaccine containing antigens from bovine viral respiratory pathogens to avoid interference from maternal antibody. Both groups of foals and adult horses responded to the vaccine with a significant increase in vaccine-specific IgGa and IgG(T) concentrations. In contrast, only adult horses and 3-month-old foals mounted significant vaccine-specific total IgG, IgGb, and IgM responses. Vaccine-specific concentrations of IgM and IgG(T) were significantly different between all groups, with the highest concentrations occurring in adult horses, followed by 3-month-old foals and, finally, 3-day-old foals. Only the adult horses mounted significant vaccine-specific lymphoproliferative responses. Baseline gamma interferon (IFN-γ) and interleukin-4 (IL-4) concentrations were significantly lower in 3-day-old foals than in adult horses. Vaccination resulted in a significant decrease in IFN-γ concentrations in adult horses and a significant decrease in IL-4 concentrations in 3-day-old foals. After vaccination, the ratio of IFN-γ/IL-4 in both groups of foals was significantly higher than that in adult horses. The results of this study indicate that the humoral and lymphoproliferative immune responses to this killed adjuvanted vaccine are modest in newborn foals. Although immune responses improve with age, 3-month-old foals do not respond with the same magnitude as adult horses.

Avian influenza (AI) is an important infectious disease in both animals and humans. Since the late 1990s, H9N2 low-pathogenicity avian influenza (LPAI) virus (LPAIV) has been reported from South Asia, South Africa, Europe, and the Middle East (1, 5, 16, 21, 23, 26), and infection has become increasingly prevalent in domestic poultry (16). H9N2 LPAI virus can be transmitted from poultry to mammalian species, including humans, by direct and indirect contact (4, 25). Therefore, the H9N2 LPAI virus is considered an important candidate source of future human pandemic influenza. In South Korea, the first H9N2 LPAI outbreak in chickens occurred in 1996 and caused slight to moderate mortality (5 to 30%) with apparent clinical signs of depression, edema in the head of the chicken, drop in egg production, and cyanosis in the comb and legs of the chicken (15, 20). H9N2 LPAI reappeared in late 1999 and has become prevalent in South Korea and has caused appreciable economic losses in the poultry industry. Vaccination is being considered more commonly as an alternative control measure to prevent avian influenza, although it can affect serologic surveillance for H9N2 LPAI infection and negatively affect international trade. H9N2 LPAI vaccination in several countries has been a promising control measure (15, 17, 21, 34); inactivated vaccines prevent clinical signs and increase resistance to infection despite the risk of masking circulation due to the shedding of field virus, if vaccinated poultry become infected with a sufficiently high dose of virus (6, 29, 30). In South Korea, an H9N2 inactivated vaccine consisting of inactivated-virus whole-virus vaccine (in oil emulsion) has been used since March 2007 in an effort to combat the widespread distribution of H9N2 LPAI since 2004, with increasing prevalence of clinical signs and infection-related mortality (7). However, an important limitation in the use of homologous inactivated vaccine is that vaccinated chickens cannot be differentiated from naturally infected chickens using serologic tests such as the agar gel immunodiffusion test (AGP), commercial enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI) tests (29). Strategies of differentiation of infected from vaccinated animals (DIVA) that have been proposed include the use of unvaccinated sentinel birds, subunit vaccines, vaccination with inactivated virus and a homologous hemagglutinin (HA) to the circulating field virus but a heterologous neuraminidase (NA) and the measurement of the serologic response to nonstructural protein 1 (NS1) (28). In South Korea, unvaccinated sentinel chickens have been placed in vaccinated flocks to determine whether flocks have been exposed to H9N2 LPAI. Although sentinels are thought to provide a sensitive measure of infection of a vaccinated flock, this strategy carries practical difficulties; the sentinel poultry must be marked, and these naive poultry may increase the risk of infection of the flock (28). Therefore, development of a diagnostic method(s) in the DIVA strategy for control of H9N2 LPAI is needed for use of the H9N2 LPAI vaccine. Recently, an alternative to DIVA strategy was reported using the measurement of a differential immune response to the extracellular domain of the third integral membrane protein (M2 protein) of the influenza virus (14). The M2e protein (extracellular domain of M2), like NS1, is also associated with actively replicating virus which is exuberantly synthesized in infected cells but is not incorporated into the infectious viral particle, and M2 protein shares with NS1 protein the potential for elaborated DIVA testing, due to differential epitope density on the surfaces of infected cells and on infectious viral particles (10, 18, 22, 35). This M2 protein consists of 97 amino acids (aa), and 24 N-terminal amino acids (M2e) are exposed outside the membrane surface (9); this M2e region is highly conserved in all human, swine, and avian influenza A viruses, with host restriction specificities (18). The main goal of the present study was to evaluate an ELISA using the extracellular domain of the M2 protein (M2e) of H9N2 LPAI virus (H9M2e ELISA) on the basis of the production of anti-M2e antibodies readily detectable in sera from infected chickens in the differentiation of H9N2 LPAIV-vaccinated chickens from infected chickens with the aim of further field application and, ultimately, replacement of the sentinel strategy used in vaccinated flocks in South Korea.

Control of blood-borne infections is dependent on antigen-specific effector and memory T cells and high-affinity IgG responses. In chronic infections characterized by a high antigen load, it has been shown that antigen-specific T and B cells are vulnerable to downregulation and apoptosis. Anaplasma marginale is a persistent infection of cattle characterized by acute and chronic high-load bacteremia. We previously showed that CD4 + T cells primed by immunization with an A. marginale outer membrane protein were rapidly deleted following infection. Furthermore, peripheral blood T cell responses to bacteria were not observed after acute infection was controlled, suggesting dysfunctional T cell priming to other A. marginale antigens. The current study more closely investigated the kinetics of A. marginale -specific CD4 + T cell responses primed during infection. Frequent sampling of peripheral blood and spleens revealed that antigen-specific CD4 + T cell responses were first detected at 5 to 7 weeks, but the responses were sporadic and transient thereafter. A similar pattern was observed in animals sampled weekly for nearly 1 year. Paradoxically, by 2 weeks of infection, cattle had developed high titers of A. marginale -specific IgG, which remained high throughout persistent infection. This dysfunctional CD4 + T cell response to infection is consistent with continual downregulation or deletion of newly primed effector T cells, similar to what was observed for immunization-induced T cells following A. marginale infection. The failure to establish a strong memory T cell response during A. marginale infection likely contributes to bacterial persistence.

Streptococcus equi subsp. zooepidemicus has been linked to cases of acute fatal pneumonia in dogs in several countries. Outbreaks can occur in kenneled dog populations and result in significant levels of morbidity and mortality. This highly contagious disease is characterized by the sudden onset of clinical signs, including pyrexia, dyspnea, and hemorrhagic nasal discharge. The pathogenesis of S. equi subsp. zooepidemicus infection in dogs is poorly understood. This study systematically characterized the histopathological changes in the lungs of 39 dogs from a large rehoming shelter in London, United Kingdom; the dogs were infected with S. equi subsp. zooepidemicus . An objective scoring system demonstrated that S. equi subsp. zooepidemicus caused pneumonia in 26/39 (66.7%) dogs, and most of these dogs (17/26 [65.4%]) were classified as severe fibrino-suppurative, necrotizing, and hemorrhagic. Three recently described superantigen genes ( szeF , szeN , and szeP ) were detected by PCR in 17/47 (36.2%) of the S. equi subsp. zooepidemicus isolates; however, there was no association between the presence of these genes and the histopathological score. The lungs of S. equi subsp. zooepidemicus -infected dogs with severe respiratory signs and lung pathology did however have significantly higher mRNA levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) than in uninfected controls, suggesting a role for an exuberant host immune response in the pathogenesis of this disease.

In developing countries, the conventional test and slaughter strategy for the control of bovine tuberculosis is prohibitively expensive, and alternative control methods such as vaccination are urgently required. In this study, the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against bovine tuberculosis (bTB) was evaluated in Holstein calves under field conditions in Ethiopia. Thirteen neonatally vaccinated and 14 control calves were exposed for 10 to 23 months to skin test reactor cows. Gamma interferon (IFN-γ) testing, comparative intradermal tuberculin testing, postmortem examination, and bacteriological culture were used for the evaluation of BCG efficacy. The overall mean pathology score was significantly ( P < 0.05) higher in control calves than in vaccinated calves. Culture positivity for Mycobacterium bovis was higher in the control calves than in the vaccinated calves, and significantly more BCG-vaccinated animals would have passed a standard meat inspection ( P = 0.021). Overall, the protective efficacy of BCG was between 56% and 68%, depending on the parameters selected. Moreover, by measuring gamma interferon responses to the antigens ESAT-6 and CFP-10, which are present in M. bovis but absent from BCG, throughout the experiment, we were able to distinguish between vaccinated animals that were protected against bTB and those animals that were not protected. In conclusion, the present trial demonstrated an encouraging protective effect of BCG against bTB in a natural transmission setting in Ethiopia.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces both humoral and cellular immune responses. In this study, we investigated the changes in cytokine levels in peripheral blood between the highly pathogenic PRRSV HuN4 strain and its derivative strain HuN4-F112 obtained by serial propagation in MARC145 cells to 112 passages. The results demonstrated that pigs infected with HuN4 showed a loss of appetite, decrease in body weight, raised body temperature, and respiratory symptoms, along with interstitial pneumonia lesions. The PRRSV amounts in the pigs infected with HuN4 were 10 5 to 10 9 copies/ml in the blood and 10 10 to 10 11 copies/g in the lung tissues, whereas the virus amounts with HuN4-F112 were 10 2.15 to 10 3.13 copies/ml in the blood and 10 3.0 to 10 3.6 copies/g in the lungs. Moreover, the levels of interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α), and alpha interferon (IFN-α) in peripheral blood were upregulated 7 days postinoculation with HuN4, which was earlier than in the HuN4-F112 group. Furthermore, cytokine levels in the pigs infected with HuN4 returned to normal on the 21st day postinoculation, while the levels in those infected with HuN4-F112 continued to increase. These results demonstrated that the pigs infected with the highly pathogenic PRRSV HuN4 strain generated earlier and higher levels of inflammatory cytokines, and the results also indicated that HuN4 may aggravate inflammation and damage tissues and organs. The low-pathogenic PRRSV HuN4-F112 strain induced lower levels of inflammatory cytokines, which may enhance the immune responses against the infection.

A panel of monoclonal antibodies (MAbs) was generated from mice immunized with binary ethylenimine (BEI)-inactivated H7N1 (A/TK/ON/18-2/00) virus. Using a dot blot assay, six of seven MAbs reacted with viruses of the H7 subtype, but not with any of the other 15 hemagglutinin (HA) subtypes tested. Four of the seven MAbs reacted with 14 different H7 isolates, indicating that the MAbs binding epitopes are conserved among viruses of the H7 subtype. The binding epitopes of all seven MAbs were conformational and reacted with the HA1 fraction of the HA protein in Western blots under nonreducing conditions. Applications of these MAbs in the development of rapid tests for H7 subtype viruses were evaluated. The MAbs demonstrated reactivity with AI virus H7 antigen in immunofluorescence and immunohistochemistry assays. Monoclonal antibody 3 showed a very strong immunostaining in the formalin-fixed and paraffin-embedded tissue from the H7N3 virus-infected chicken. A double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed using two of the MAbs. The DAS ELISA specifically detected all H7 strains tested in this study. A competitive ELISA (cELISA) for the detection of H7-specific antibodies was evaluated using one MAb and BEI-inactivated H7N1 virus as the antigen. All infected birds showed positive antibody responses at 7 days postinfection. The sensitivity of this cELISA was comparable with that of an influenza A nucleoprotein-based cELISA. This panel of MAbs is valuable in the development of various immunoassays.

Lactobacillus acidophilus , Lactobacillus reuteri , and Lactobacillus salivarius are all normal residents of the chicken gastrointestinal tract. Given the interest in using probiotic bacteria in chicken production and the important role of the microbiota in the development and regulation of the host immune system, the objective of the current study was to examine the differential effects of these bacteria on cytokine gene expression profiles of lymphoid tissue cells. Mononuclear cells isolated from cecal tonsils and spleens of chickens were cocultured with one of the three live bacteria, and gene expression was analyzed via real-time quantitative PCR. All three lactobacilli induced significantly more interleukin 1β (IL-1β) expression in spleen cells than in cecal tonsil cells, indicating a more inflammatory response in the spleen than in cecal tonsils. In cecal tonsil cells, substantial differences were found among strains in the capacity to induce IL-12p40, IL-10, IL-18, transforming growth factor β4 (TGF-β4), and gamma interferon (IFN-γ). In conclusion, we demonstrated that L. acidophilus is more effective at inducing T-helper-1 cytokines while L. salivarius induces a more anti-inflammatory response.

The aim of the present study was to characterize the antigenic specificity of the humoral immune response made by cats infected with the feline hemoplasma,Mycoplasma haemofelis.A crudeM. haemofelisantigen preparation was prepared from red blood cells (RBCs) collected from a cat at the time of a high level of bacteremia. Plasma samples were collected from six cats before and after experimental infection withM. haemofelis, with regular sampling being performed from 15 to 149 or 153 days postinfection (dpi). Preinfection RBC membrane ghosts were prepared from these six cats and used to identify erythrocyte proteins that may have contaminated theM. haemofelisantigen preparation. TheM. haemofelisantigen preparation comprised 11 protein bands. The immunodominant bands on Western blotting with infected cat plasma had molecular masses of 78, 68, 60, 48, and 38 kDa. Most cats (n= 5) had plasma antibody that reacted with at least one band (always including the one of 68 kDa) at 15 dpi, and all cats were seroreactive by 29 dpi. The maximum number of antibodies from an individual animal specific for an antigen was identified in plasma collected from 57 to 99 dpi. Contamination of theM. haemofelisantigen preparation with RBC membrane proteins was observed. The contaminating RBC proteins had molecular masses of from 71 to 72 kDa (consistent with band 4.2) and 261 and 238 kDa (consistent with spectrin), and these were recognized by all plasma samples. A range ofM. haemofelisantigens is recognized by cats infected experimentally with the organism. These represent possible targets for immunoassays, but care must be taken to prevent false-positive results due to host protein contamination.