Assessment of the safety and immunogenicity of Rhodococcus equi-secreted proteins combined with either a liquid nanoparticle (IMS 3012) or a polymeric (PET GEL A) water-based adjuvant in adult horses and foals—Identification of promising new candidate antigens

Rhodococcus equi is the most common infectious cause of mortality in foals between 1 and 6 months of age. Because of an increase in the number of antibiotic-resistant strains, the optimization of a prophylactic strategy is a key factor in the comprehensive management of R. equi pneumonia.The objectives of this study were to assess the safety and immunogenicity of R. equi-secreted proteins (ReSP) co-administered with either the nanoparticular adjuvant Montanide? IMS 3012 VG, or a new polymeric adjuvant Montanide? PET GEL A, and to further investigate the most immunogenic proteins for subsequent immunization/challenge experiments in the development of a vaccine against rhodoccocal pneumonia. The approach involved two phases. The first phase aimed to investigate the safety of vaccination in six adult horses. The second phase aimed to determine the safety and immunogenicity of vaccination in twelve 3-week-old foals.We set out to develop a method based on ultrasound measurements for safety assessment in adult horses in order to evaluate any in situ changes at the injection site, in the skin or the underlying muscle, with quantitative and qualitative data revealing that administration of ReSP combined with the Pet Gel A adjuvant led to an increase in local inflammation, associated with 4- to 7-fold higher levels of anti-R. equi IgGa, IgGb and IgGT, compared to administration of ReSP associated with IMS 3012 adjuvant, but without any impact on animal demeanor. Investigations were then performed in foals with serological and clinical follow-up until 6 months of age. Interestingly, we observed in foals a much lower incidence of adverse local tissue reactions at the injection site than in adult horses, with transient and moderate swelling for the group that received ReSP combined with Pet Gel A. Immunized foals with Pet Gel A adjuvant exhibited a similar response in both IgGa and IgGT levels, but a lower response in IgGb levels, compared to adult horses, with a subisotype profile that may however reflect a bias favorable to R. equi resistance. From the crude extract of secreted proteins, dot-blot screening enabled identification of cholesterol oxidase, mycolyl transferase 3, and PSP (probable secreted protein) as the most immunogenic candidates. Taken together, these results are encouraging in developing a vaccine for foals.     

Cytokine expression by neutrophils of adult horses stimulated with virulent and avirulent Rhodococcus equi in vitro

Rhodococcus equi is an intracellular pathogen of macrophages that causes rhodococcal pneumonia in foals and immunocompromised people. Evidence exists that neutrophils play a vital role in resistance to infection with R. equi; however, the means by which neutrophils exert their effects have not been clearly defined. In addition to directly killing bacteria, neutrophils also may exert a protective effect by linking innate and adaptive immune responses. In the present study we evaluated the cytokine expression profiles of adult equine neutrophils in response to stimulation with isogenic strains of virulent and avirulent R. equi in vitro. After 2 and 4 h incubation with virulent or avirulent R. equi, adult equine neutrophils expressed significantly (P < 0.05) greater tumor necrosis factor alpha (TNFα), interleukin (IL)-12p40, IL-6, IL-8 and IL-23p19 mRNA, but not interferon gamma (IFNγ) or IL-12p35 mRNA than unstimulated neutrophils. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA than avirulent R. equi. These results demonstrate that R. equi-stimulated neutrophils are a source of many proinflammatory cytokines. Furthermore, these results suggest that IL-23 may be preferentially expressed over IL-12 in response to exposure with R. equi, and that this response may be more strongly induced by virulent R. equi than avirulent R. equi. Collectively, the data presented herein suggest a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.     

Mice immune responses to Brucella abortus heat shock proteins. Use of baculovirus recombinant-expressing whole insect cells,purified Brucella abortus recombinant proteins,and a vaccinia virus recombinant as immunogens

Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.     

Identification of a novel protective antigen, 3-oxoacyl-[acyl-carrier-protein] synthase II of Streptococcus equi ssp. zooepidemicus which confers protective effects

Streptococcus equi ssp. zooepidemicus (SEZ) is an important swine pathogen and responsible for a wide variety of infections in many animal species. FabF was a novel protein identified in the previous study. However, its protective efficacy remained to be evaluated. In this study, recombinant fabF of SEZ was expressed and showed a strong immunoreactivity with mini-pig convalescent sera. Study in mice revealed that the recombinant protein induced a marked antibody response and protected 80% of mice against SEZ infection. The hyperimmune sera against fabF could efficiently kill the bacteria in the phagocytosis test. In addition, it was also found that anti- fabF antibodies can significantly inhibit the formation of SEZ biofilm. These study suggest that fabF may represent immunogens of interest for vaccine development against SEZ infection.     

Expression of 4 truncated fragments of Pasteurella multocida toxin and their immunogenicity

Pasteurella multocida toxin (PMT) is a poor antigen that becomes more immunogenic after its native structure has been destroyed. In contrast, partially truncated PMT proteins, which are predicted to be good antigens when used as a vaccine, might be used to improve the control of atrophic rhinitis in pigs. In this study, 4 truncated PMT fragments were expressed in Escherichia coli, and those 4 fragments were inoculated into mice to produce the polyclonal antibodies. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that #1 and #4 fragments were the most immunogenic. Immunized mice were subsequently challenged intraperitoneally with P. multocida type D. Five of the eight #1 fragment-immunized mice showed some protection against death and bacterial clearance. Pigs immunized with #1 fragment produced no or mild atrophic rhinitis (turbinate conchal score) after challenge, suggesting that this #1 fragment could be a good candidate for a subunit recombinant-type vaccine.     

Equine ehrlichiosis: A comparison between E. equi and other pathogenic species of Ehrlichia

Ehrlichia equi, etiologic agent of equine ehrlichiosis, is a rickettsia which morphologically closely resembles the agents of bovine petechial fever, tick-borne fever, and canine ehrlichiosis (tropical canine pancytopenia). Natural infections of E. equi have been reported only in horses; however, the experimental host range of E. equi has been broadened to include burros, sheep, goats, dogs, cats, monkeys and baboons. Infection of primates indicates that E. equi may be a zoonotic agent. An indirect fluorescent antibody test employing E. equi-infected granulocytes as antigen has been developed and used to show that infected horses develop a prolonged antibody response to E. equi. Cell-mediated immune responses measured by the leukocyte migration inhibition test were also detected in horses recovered from acute illness. Protective immunity in horses, monkeys and baboons to reinfection is long-lasting. In contrast to the blood of dogs recovered from clinical E. canis infection, the blood of horses and dogs recovered from clinical infection with E. equi is not infectious for susceptible animals. Infection of dogs with E. equi does not provide protection against subsequent infection with E. canis.     

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.     

Evaluating the immune responses of mice to subcutaneous immunization with Helicobacter pylori urease B subunit

Background

Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural subunits of urease, was expressed in E. coli BL21 (DE3) strain. The objective of this study was to evaluate the effects of Helicobacter pylori urease B subunit on the immune responses in mice by subcutaneous immunization.

Methods

The mice were immunized and boosted with Helicobacter pylori urease B subunit antigen subcutaneously three times with 2-wk intervals between the immunizations and boosters. The mice in the control group were immunized with PBS. The adjuvant group received PBS containing complete/incomplete freund’s adjuvant identical to antigen group without Helicobacter pylori urease B subunit antigen. Four weeks after the final booster, all the mice were sacrificed. Blood was collected on d 0, 14, 28 and 56 before immunization, booster and sacrifice, respectively. Immediately after sacrifice, gastric liquid and spleen were collected for antibody and cytokine analyses.

Results

Urease B subunit increased the concentrations of serum and gastric anti-urease B antigen specific IgG, and the levels of interleukin-4 and interferon-γ in splenocytes of the mice (P < 0.05).

Conclusions

This study demonstrated that recombinant urease B subunit can induce systemic and local immune responses in mice by subcutaneous immunization, which might be used as the effective component of vaccine against Helicobacter pylori.     

Serological epidemiological surveillance for vapN-harboring Rhodococcus equi infection in goats

Rhodococcus equi causes suppurative pneumonia in foals aged 1–3 months; moreover, it has emerged as a pathogenic cause of zoonotic diseases. After the initial report of the ruminant-pathogenic factor VapN encoded by the novel virulence plasmid pVAPN, several reports have described ruminant infections caused by vapN-harboring R. equi. Herein, we conducted a serological epidemiological surveillance in goats at a breeding farm (Farm A) and characterized the vapN-harboring R. equi isolates from this farm. First, we established a simple screening enzyme-linked immunosorbent assay (ELISA) using recombinant glutathione S-transferase–tagged VapN as an immobilized antigen. This method revealed that the VapN antibody titers were elevated in 12 of 42 goats. Subsequently, we attempted to isolate R. equi from the goat feces and soil of Farm A. choE⁺/vapN⁺ R. equi was isolated from the feces of Goat No. 27 and a soil sample near the shed. The pulsed-field gel electrophoresis (PFGE) patterns of five vapN-harboring R. equi strains isolated from Farm A in 2013 and 2019 were investigated and found to be the same except for the strain (OKI2019F1). However, no difference was observed in VapN expression and growth in macrophages among these vapN-harboring R. equi isolates. Our results revealed that some goats had histories of vapN-harboring R. equi infections, and two genomic types of vapN-harboring R. equi were found in isolates from Farm A. Ruminant-specific (pVAPN-carrying) R. equi might be an unrecognized pathogen in Japan and further studies are required to determine its prevalence and distribution.     

Assessment of the immunogenicity of the leptospiral LipL32, LigAni,and LigBrep recombinant proteins in the sheep model

Veterinary leptospirosis vaccines are composed of bacterins and present limitations, for example, the need for bacteriological culture and serovar-dependent immunity. Recombinant antigens represent a promising alternative. LigAni, LigBrep, and LipL32 proteins have been shown to promote a protective immune response against the homologous challenge in hamsters. Therefore, the next step is to evaluate the immunological properties of these immunogens in the actual hosts, as ruminants, which has never been performed before. The objective of this study was to evaluate the immunogenicity and potential adverse effects of the recombinant proteins LigAni, LigBrep, and LipL32 in the ovine model. For this, 16 Santa Inês sheep were allocated into three groups: two experimental (Groups A and B) and one control group (Group C). Group A was inoculated with a formulation containing the recombinant proteins in combination with the aluminum hydroxide adjuvant; Group B was inoculated with a formulation containing the recombinant proteins in combination with the Montanide adjuvant; and Group C was inoculated with adjuvants only. The results revealed that formulations containing the recombinant proteins induced total IgG seroconversion and led to a significant increase in antibody titers in the sheep model. Besides, there were no clinical changes or adverse effects. Thus, LigAni, LigBrep, and LipL32 proteins elicited a significant humoral immune response with elevated serum IgG levels, demonstrating that they possess the immunogenic and safety characteristics necessary to sustain their potential use as leptospirosis vaccines in the ruminant model.     

Characterization of the main immunogenic proteins in Brucella infection for their application in diagnosis of brucellosis

Brucellosis is an important zoonotic bacterial disease widespread in the world. The key step of control this disease is accurate diagnosis and elimination of diseased animals. The classic diagnostic methods, such as tube agglutination test, are inaccurate and nonspecific, because of cross-reaction with Yersinia enterocolitica serotype O:9. Previously, several proteins were reported as Brucella main immunogens. In this study, we used animal infection model to evaluate antibody production against OMP16, BP26, BLS, BCSP31, VirB12, SodC and GroEL proteins and investigated their application in diagnosis of brucellosis. The results showed that the BP26 and BLS are two best immunogenic proteins. In further study, we detected 44 clinical bovine sera using western blot, showing that the BP26 and BLS reacted with 30 Brucella-positive sera, but false-positive results were also shown in 14 Brucella-free sera. In an indirect ELISA assay, compared to lipopolysaccharide-based ELISA, the conformance of the BP26-based ELISA was 92.68 % in Brucella-positive sera, but only 52.94 % in Brucella-free sera. The BLS-based ELISA can hardly differentiate positive sera from negative sera. Besides, truncated fragments of the BP26 protein cannot exclude false-positive results in detection of Brucella-free sera. Altogether, although Brucella main immunogenic proteins have good reaction with Brucella-positive sera, false-positive reaction with Brucella-free sera may lead to misdiagnosis of brucellosis, suggesting that it should be more careful to use these immunogenic proteins as antigen targets to diagnosis of brucellosis.     

Frequency of Rhodococcus equi in Feces of Mares in Central Kentucky

Rhodococcus equi (R equi) pneumonia is an important cause of disease and death in foals. Feces from mares can contain R equi, including virulent R equi, and thus may act as a source of the bacteria at horse breeding farms. A previous report documented that every mare at a farm in central Kentucky shed virulent R equi in at least one of four fecal samples collected serially during the periparturient period. The objective of this study was to assess the extent to which this high prevalence of fecal shedding could be replicated at other horse breeding farms. The frequency of detection of R equi and virulent R equi in fecal samples was studied among 131 mares from 24 farms in central Kentucky. The proportions of fecal samples from mares containing R equi and virulent R equi were 95% and 76%, respectively. These findings indicate that R equi and virulent R equi may be isolated with high frequency from feces of mares at breeding farms in central Kentucky, and that mares are a source of virulent R equi for the environment of their foals.     

Successful management of multiple extrapulmonary complications associated with Rhodococcus equi pneumonia in a foal

This report describes the diagnosis and successful treatment of multiple extrapulmonary sequelae of Rhodococcus equi (R. equi) pneumonia in a 3‐month‐old filly. Bilateral uveitis and hyphaema, haemolytic anaemia and polysynovitis developed in this foal and were likely due to immune‐mediated mechanisms. The challenges associated with diagnosis and treatments of these extrapulmonary disorders are discussed. The filly was treated initially with clarithromycin and rifampin; however, a blood transfusion and immunosuppressive therapy with dexamethasone were required due to progressive haemolysis and for treatment of uveitis and polysynovitis. Bilateral hyphaema was successfully treated with intracameral injections of a recombinant tissue plasminogen activator. The development of antimicrobial resistance in R. equi was an additional challenge encountered in the management of this case and emphasises the importance of culture and in vitro antimicrobial susceptibility testing of isolates from foals with R. equi pneumonia. Extrapulmonary disorders associated with R. equi pneumonia are likely underdiagnosed and associated with a poor prognosis. This case highlights the importance of thorough and ongoing diagnostic assessment of foals with R. equi pneumonia and demonstrates that a successful outcome can be achieved with appropriate and directed treatment.     

Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.     

Experimental Studies on the Pathogenesis of Corynebacterium equi Infection in Foals

Four month-old foals were infected orally with 75 mL of a suspension of 5.0 × 10⁸ Corynebacterium equi per mL. Two foals were killed after ten days and had scanty number of C. equi in the caeco-colic lymph nodes. No C. equi were recovered from the other two foals, killed 20 days after infection. No gross pathological change was detected in these four foals, although mild microscopic lesions were seen in the ileum of one foal. Results of lymphocyte blastogenesis using peripheral blood lymphocytes and C. equi antigens showed, however, that lymphocytes became sensitized to C. equi following this challenge.

In a second experiment four month-old foals were given orally the same dose of organisms but on five consecutive days. Two foals were killed ten days after infection and showed mild histological changes in the large bowel mucosa and C. equi could be recovered from all intestinal lymph nodes cultured. In one of these foals moderate numbers of C. equi were present in the bronchial lymph node. Of the other two foals, one died after 22 days with severe ulcerative enterocolitis and intestinal lymphadenitis. Only one small pulmonary abscess was detected despite large numbers of C. equi in the lungs. The other foal developed similar intestinal changes and was euthanized 25 days after infection. No C. equi were detected in the lungs or bronchial lymph node. Lymphocyte blastogenesis in these animals showed a rapid rise in response to C. equi antigens.

These studies suggest that C. equi pneumonia in foals does not always arise from an intestinal infection, that minor intestinal infection causes a cellular immune response and that massive exposure of the bowel over a sustained period is necessary to induce intestinal lesions.

     

Serological and molecular detection of Theileria equi in sport horses of northeastern Brazil

Theileriosis is a worldwide protozoal tick-borne disease caused by Theileria equi, which may produce a variety of clinical signs and turn infected horses into lifetime carriers. This study has aimed to perform a serological and molecular detection of T. equi and associated factors in sports horses from six areas of northeastern Brazil. In overall, 59.6% horses were positive by indirect immunofluorescence assay and 50.4% by polymerase chain reaction. No significant association was found when presence of ticks, age, gender, anemia or total plasma proteins was analyzed with seropositivity and molecular techniques. Although a significant association of infection was found in two cities. Thus, local risk factors other than presence of ticks, horse age, gender, anemia and total plasmatic proteins may dictate prevalence of T. equi infection in sports horses, even in highly endemic areas with no control of infection prior to horse competitions.     

Outer membrane proteins of bovine Pasteurella multocida serogroup A isolates

The outer membrane proteins (OMPs) of P. multocida serotypes A3 (7 isolates), A4 (2 isolates), A3,4 and A2 (one isolate each) obtained from pneumonic cattle (10 isolates) and from one pig isolate were investigated to identify potential immunogens. SDS-PAGE of P. multocida OM isolated by SDG centrifugation of spheroplasts revealed eight major OMPs. Outer membranes isolated by sarcosyl extraction or SDG had similar protein composition on Coomassie blue-stained SDS-PA gel and on immunoblots. Two major OMPs (M_rs of 35 and 46 kDa at 100°C) demonstrated heat modifiability with apparent M_rs of 30 and 34 kDa at 37°C, respectively. The N-terminal aa sequences of these heat modifiable proteins revealed homology with E. coli OmpA and Hib P1 proteins, respectively. Protease treatment of whole cells followed by western immunoblots using bovine convalescent sera identified several immunogenic, surface-exposed and conserved OMPs among the eleven P. multocida isolates examined. The whole organism SDS-PAGE profiles of the eleven P. multocida isolates differed such that six patterns were seen. These patterns could potentially be used as a typing system for P. multocida bovine isolates based on the molecular weights of whole cell proteins. The above observations have potentially important implications relative to the immunity to infection.