The Evidence Base for Prevention of Infectious Bovine Keratoconjunctivitis Through Vaccination.

Pili and cytotoxins are important virulence factors and antigens for Moraxella spp. Local and systemic immunity may play a role in the body's response to infectious bovine keratoconjunctivitis (IBK). No evidence exists that eliminating the carrier state for IBK is possible or beneficial. Evidence for efficacious transfer of passive immunity from dams to calves is conflicting. Autogenous vaccines and commercial vaccines for putative pathogens for IBK have not yet shown efficacy in blinded randomized field trials. Study design features, such as randomization, blinding, diagnostic criteria, and use of a placebo, reduce the risk of bias in vaccine studies for IBK.

Systemic and ocular immune responses in cattle following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid.

OBJECTIVE To evaluate changes in systemic and ocular antibody responses of steers following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin (MbxA). ANIMALS 13 Angus steers with ages ranging from 318 to 389 days and weights ranging from 352 to 437 kg. PROCEDURES Steers were assigned to receive 500 µg of a precipitated (MbxA-P; n = 5) or partially solubilized (MbxA-S; 5) recombinant MbxA subunit adjuvanted with polyacrylic acid. A control group (n = 3) received the adjuvant alone. Each steer received the assigned treatment (1 mL/nostril) on days 0 and 28. Serum and tear samples were collected on days 0 (before vaccination), 14, 28, 42, and 55. Changes in MbxA-neutralizing antibody titers and MbxA-specific IgG concentrations in serum and tears and changes in MbxA-specific IgA concentrations in tears were measured. RESULTS Mean fold changes in MbxA-specific IgG concentration in serum and tears and MbxA-neutralizing antibody titer in tears for the MbxA-P group were significantly greater than those for the MbxA-S and control groups. Mean serum MbxA-neutralizing antibody titer did not differ among the 3 groups. Although the mean fold change in tear MbxA-specific IgA concentration differed significantly among the groups in the overall analysis, post hoc comparisons failed to identify any significant pairwise differences. CONCLUSIONS AND CLINICAL RELEVANCE Systemic and ocular immune responses induced by intranasal administration of the MbxA-P vaccine were superior to those induced by the MbxA-S vaccine. Additional research is necessary to determine whether the MbxA-P vaccine can prevent naturally occurring infectious bovine keratoconjunctivitis.

Is visual function affected in severe ocular allergies?

PURPOSE OF REVIEW: The purpose of the present review is to provide an overview on the clinical features and long-term complications of severe ocular allergies, with a special focus on the corneal involvement observed in these diseases, which may impair vision permanently. Furthermore, different treatment options are discussed based on the sight-threatening effects of corticosteroid use. RECENT FINDINGS: Recent data on the demographics, clinical and immunologic features of vernal and atopic keratoconjunctivitis are presented and discussed, and the evidence of a lack of literature addressing the issue of visual outcome in allergic diseases is underlined. The efficacy and possible long-term complications of their treatments are described, including visual impairment. SUMMARY: Diagnosis and treatment of patients with severe ocular allergy is a challenge for ophthalmologists due to the long-term corneal complications and the potential side-effects of topical corticosteroids that may induce cataract formation and glaucoma, and impair vision permanently. The review describes old and new concepts of management and possible complications such as severe, vision-threatening forms of allergic ocular disease. An update on their management and long-term complications may help clinicians to establish a common agreement on treatment options and researchers to design future studies based on similar outcomes, including visual acuity.

Bactericidal activity of lachrymal secretion and complement system in copper deficient bovines.

Copper deficiency in humans and animals has been related to increased susceptibility to infections. Neutrophils are one of the most studied components of the immune response; however, to the best of our knowledge, other defenses of the innate immune system have not been analyzed in copper-deficient animals. Our previous studies in copper-deficient bovines have shown increased susceptibility to infectious keratoconjunctivitis, an ocular disease caused by Moraxella bovis. The objective of this work was to evaluate the bactericidal activity of lachrymal secretion and complement system, two main mechanisms of the innate immune response against M. bovis, in copper-deficient cattle. Our results indicate that copper deficiency has no effect on bactericidal activity of complement system and lachrymal secretion against M. bovis in calves. Other components of local and systemic ocular defense mechanisms that could explain the increased susceptibility to infectious keratoconjunctivitis observed in copper-deficient bovines should be investigated.

Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS: 107 beef steers. PROCEDURES: 2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined. RESULTS: No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non-IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves. CONCLUSIONS AND CLINICAL RELEVANCE: The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine: evaluation of immunity and pathology.

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Evaluation of cytokines as adjuvants of infectious bovine keratoconjunctivitis vaccines.

In this work two cytokines were used in combination with inactivated bacteria (bacterin) to test the bovine conjuctival immune response to the pathogen Moraxella bovis, the causative agent of Infectious bovine keratoconjunctivitis (IBK). Treatments using the bacterin vaccine combined with interleukin-2 and interferon-α as adjuvants (Group A), the bacterin vaccine only (Group B), and controls without treatment (Group C), were applied by ocular spraying to evaluate the local immune response in the corneal structure of cattle experimentally infected with M. bovis. Six weeks after infection, 14 out of a total of 34 animals presented different corneal lesions; 9 corresponding to the control group C, 4 to the group B and only one to the group A. According to the clinical manifestations, a numeric score was calculated. Control animals presented the highest score value (12 points), followed by group B (7.5 points) and group A (1 point). These results suggest that the addition of cytokines to M. bovis treatments can reduce not only eye injuries caused by IBK but also the number of diseased animals.

Dynamics of Moraxella bovis infection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves.

Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.

Association of toll-like receptor four single nucleotide polymorphisms with incidence of infectious bovine keratoconjunctivitis (IBK) in cattle.

Toll-like receptor 4 (TLR4) is a receptor protein that binds pathogen ligands, which are mainly associated with Gram-negative bacteria. The objective of this study was to investigate the association of nucleotide polymorphisms in TLR4 with infectious bovine keratoconjunctivitis (IBK), or pinkeye, incidence in American Angus cattle. Animals with previously calculated breeding values for IBK susceptibility were used to identify two SNPs in TLR4; Int1 (A/G) in intron1 (-26 Ex2 position) and Ex3 (C/T) in exon3 (1,678 position). To investigate the possible role of these SNPs in IBK susceptibility, the disease incidence information was collected on 370 calves raised in Iowa at two time points-June or August (disease season) and October (at weaning) and genotyped using PCR-RFLP protocols. In statistical models including year, pasture management group, and SNP, the Int1 SNP had a significant effect on IBK infection rates both in-season (P < 0.05) and at weaning (P < 0.01), whereas the Ex3 SNP was not significant (P > 0.79) at either time point. Furthermore, the Int1 SNP alone could account for 2.1% of phenotypic variation in IBK infection during the disease season and 3.0% of phenotypic variation in IBK infection at the time of weaning. These data indicate that there is a relationship between Int1 genotype and the rate of IBK infection in American Angus cattle.

A randomized and blinded field trial to assess the efficacy of an autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves.

A randomized and blinded 2-arm parallel trial was conducted to assess the efficacy of an autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves. The trial was managed between May and November 2008 on university owned farms in Iowa and Wisconsin. The vaccine at Iowa contained Moraxella bovoculi (M. bovoculi) while the organism used in the Wisconsin herds vaccine was Branhemella ovis (B. ovis renamed M. ovis). Calves born between January and May 2008 without visible corneal lesions were randomized to receive an autogenous vaccine or placebo vaccine using a computer generated sequence. Two subcutaneous doses were administered 21-28 days apart. Allocation to treatment was concealed using bottles marked A or B. Staff were blind to the treatment allocation. The primary outcome was IBK cumulative incidence over the study period. The secondary outcome was weaning weight. Only the Iowa herd met the criteria for an "at-risk" herd i.e. >15% IBK in unvaccinated calves and M. bovoculi isolation from IBK cases. Analysis was "per-protocol". The cumulative incidence of IBK was 47/105 in vaccinated calves and 49/109 in unvaccinated calves (unadjusted odds ratio=0.99, 95% CI: 0.58-1.70). Weight at weaning did not differ between the vaccinated cohort 148kg (SD: +/-27) and unvaccinated cohort 146kg (SD: +/-26) (unadjusted beta=1.5 and 95% CI: -5.5 to 8.6). Results indicate that the autogenous vaccine was ineffective in this study population.

Development of prophylactic recombinant HPV58-attenuated Shigella live vector vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model.

To develop a prophylactic recombinant HPV58L1-attenuated Shigella live vector vaccine and evaluate its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model, the HPV58L1 gene was cloned into vector pUCmt, and then subcloned into the suicide vector pCVD442. The recombinant plasmid pCVD442-HPV58L1 was introduced into attenuated Shigella (sf301:deltavirG) with the helper plasmid PRK2013 by filter mating. The positive colonies were harvested and confirmed by polymerase chain reaction. The expression of the HPV58L1 protein with a molecular weight of 60 kDa was confirmed by western blot. The ability of the interested protein to self-assemble into virus-like particles was identified by transmission electron microscope, and murine erythrocyte hemagglutination assay. The guinea pig keratoconjunctivitis model was used to evaluate the protective efficacy and immunogenicity of the vaccine. Animal experiments showed that there was no keratoconjunctivitis occurred in the immunized group (HPV58-attenuated Shigella), and the serum levels of anti-HPV58L1-IgG and -IgA were obviously increased (P < 0.05), but the anti-sf301 LPS-IgG just slightly increased (P > 0.05). Enzymelinked immunosorbent spot assay showed that HPV58L1-specific IgA-antibody-secreting cells (ASC) and IgG-ASC of spleen and lymph nodes were also obviously increased (P < 0.01). In this study, a recombinant HPV58L1-attenuated Shigella live vector vaccine was successfully constructed, and it could induce strong humoral immune responses in the immunized animals, and induce protective antibody production.

Serologic cross-reactivity of Australian Moraxella bovis to vaccinal bacterin strains as determined by competitive ELISA.

OBJECTIVE: To conduct a serologic survey and define pili antigenic variability via the serologic cross-reactivity of Moraxella bovis isolates from naturally occurring infectious bovine keratoconjunctivitis (IBK) outbreaks in Australia. This project applies to the development of an M bovis pili-based vaccine targeting Australian strains originating from intensive cattle producing regions. PROCEDURE: Ocular swabs were collected from cattle affected with clinical signs of IBK from 25 veterinary practices. Standard criteria were used to identify 70 M bovis. Pure, piliated isolates were evaluated with a modified competitive enzyme-linked immunosorbent assay (ELISA) for cell-bound M bovis pili to determine their serologic cross-reactivity with pili of vaccinal bacterin strains EPP63, FLA64, and SAH 38. RESULTS: Sixty-four percent (45/70) of M bovis isolates demonstrated homologous pili antigens to a vaccinal strain. M bovis isolates homologous to one of the three vaccinal strains were obtained in 77% (34/44) of IBK outbreaks sampled. No IBK outbreak had isolates homologous to more than one vaccinal strain; however, 29% (10/34) of outbreaks with a cross-reacting strain had non-cross-reacting strains as well. CONCLUSION: The similar prevalence of pilus antigen homology to strain FLA64 was observed with isolates derived from NSW, Tasmania, and Victoria, compared with results of prior smaller serologic studies, suggests that the common pilus antigens in M bovis within Australia have been relatively stable over the last 20 years. The prevalence of a limited number of pilus antigens in M bovis suggest that the application of a vaccine containing the bacterial strains EPP63, FLA64, and SAH38 may provide a useful management tool for reducing production losses associated with IBK in Australia.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

The efficacy of a recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK) was evaluated in a randomized, blinded, controlled field trial. Ninety-three cross bred beef calves were vaccinated with either saline, ISCOM matrix (adjuvant control), or a recombinant M. bovis cytotoxin carboxy terminus peptide plus ISCOM matrix and boostered 21 days later. Ocular examinations were performed once weekly for 20 weeks. At week 12, the cumulative proportion of calves with ulcerated eyes in the recombinant vaccine group was significantly lower than in the saline control group. Throughout the 20 week trial, the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine group. By week 7, nonulcerated calves in the recombinant vaccine group had significantly higher changes in serum neutralizing titers and cytotoxin specific to total IgG ratios in serum and tears as compared to calves in the control groups. The trend for a reduced cumulative proportion of IBK in the vaccinated calves over the 20 week trial suggests that a recombinant M. bovis cytotoxin vaccine may be beneficial in helping to prevent naturally occurring IBK.

Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili.

Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry. M. bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease. The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components. In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M. bovis cell surface. A sequential competitive bact-ELISA was developed using highly piliated M. bovis cells as antigen. Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M. bovis. This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M. bovis cells, and represents an important tool for cellular piliation monitoring daburing M. bovis cells production in stirred bioreactors.

Mycoplasma conjunctivae infection is self-maintained in the Swiss domestic sheep population.

Bacteriological and serological investigations were performed to assess whether the domestic sheep population is a reservoir of Mycoplasma conjunctivae in Switzerland. Among a sample of 69 sheep showing clinical signs of infectious keratoconjunctivitis (IKC) in three Swiss cantons, M. conjunctivae was identified 53 times (76.8%). A commercially prepared indirect ELISA was used to detect M. conjunctivae antibodies in 674 sera of adult sheep. We analysed a stratified random sample of 123 sheep herds from 25 out of the 26 Swiss cantons. At least one positive animal was detected in 89.4% of the herds. In positive herds (n=110), 57.1% of the individual animals tested positive. To assess the importance of sheep's age in the spread of M. conjunctivae, 209 sera of adult sheep and 93 lamb sera among eight sheep herds were analysed using the indirect ELISA. Seroprevalence in 2-6-month-old lambs was 50.5%, indicating that the IKC agent is spread in sheep flocks during raising. Lambs experimentally infected with M. conjunctivae carried the agent for 8 and 23 weeks, respectively, depending on the strain used for challenge. We conclude that the M. conjunctivae-infection is endemic and self-maintained in the domestic sheep population in Switzerland.

Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis.

The serological cross reactions between Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), and the antigenetically and phylogenetically closely related Mycoplasma ovipneumoniae, which is often found in sheep, were analysed. Cross reacting antigens were identified using sera from sheep with IKC and from sheep of herds known to be free of IKC, as well as rabbit hyperimmune serum specific to the two Mycoplasma species. Cross reactions were predominantly due to the strongly antigenic proteins of 42 kDa and 83 kDa. Serospecific antigens of M. conjunctivae could be separated from cross-reacting antigens by the extraction of Tween 20-soluble membrane proteins. The Tween 20-extracted proteins of the M. conjunctivae strain HRC/581T were used for the development of an indirect ELISA test. This ELISA test was shown to be a useful serological method for the diagnosis of M. conjunctivae infections and to identify infected sheep herds.

Effects of buprenorphine on immunogenicity and protective efficacy in the guinea pig keratoconjunctivitis model (Sereny test).

Shigellosis is a disease of global proportions, with an estimated 164.7 million episodes annually throughout the world as well as an estimated 1.1 million associated mortalities in developing countries. Due to increasing incidence, and continued emergence of multi-drug resistant strains, Shigella vaccine development is considered a top public health priority. The guinea pig keratoconjunctivitis model, the basis for the Sereny test, remains the most reliable in vivo indicator of virulence of Shigella strains and immunogenicity and protective efficacy of Shigella vaccine candidates. The model is effective in evaluating the ability of Shigella strains to invade the corneal epithelia of guinea pigs and spread to contiguous cells, with the more virulent strains causing ulcerative keratoconjunctivitis. However, analgesia is not routinely used to relieve this painful condition because of potential immunomodulation and confounding of experimental results. The objective of the study reported here was to evaluate use of buprenorphine hydrochloride as an analgesic during the Sereny test. Local and systemic immune responses were measured in guinea pigs given buprenorphine versus those responses in controls. Results of this study suggest that buprenorphine, administered at an analgesic dose of 0.05 mg/kg of body weight twice daily, can be successfully used with the model without significantly affecting immunologic evaluation of Shigella vaccine candidates. However, in buprenorphine-treated animals, there was a significant increase in the amount of mucopurulent ocular discharge, requiring frequent cleaning of the affected eyes. Additionally, animals treated with buprenorphine had significant reduction in body weight, in comparison with saline controls.

Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland.

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.