Preliminary characterisation of extracellular serine proteases of Dermatophilus congolensis isolates from cattle,sheep and horses

Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7–10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.     

A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds.     

Immunoglobulin G responses in 21 dogs with skin diseases to antigens from different isolates of Staphylococcus intermedius

The aim of this study was to characterise the immunoglobulin G (IgG) response in 21 dogs with or without pyoderma to antigens from six isolates of Staphylococcus intermedius. The staphylococcal proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred electrophoretically on to a membrane and subjected to immunoblotting with the dogs' serum. Gels containing separated proteins from the six isolates revealed 29 to 33 distinct bands with molecular weights ranging from 20 to 230 kDa. All the dogs' sera contained IgG that recognised 12 to 24 bands (mean 17), regardless of whether the dogs had pyoderma. The recognised proteins had molecular weights ranging from 20 to 198 kDa but the majority had molecular weights below 75 kDa. The most intense band in all six isolates had a molecular weight of 28 to 29 kDa. The antibody responses to the six isolates were essentially similar except that there were significantly more bands in the response to isolate 2 than to isolate 6, and occasional differences in the intensity of individual bands. All 21 dogs mounted an IgG response to multiple antigens in S intermedius, which differed only marginally between the six isolates. This lack of variation provides evidence that the host's response to different isolates of S intermedius is not a major factor in canine pyoderma.     

Antigen specificity of antibody responses to Corynebacterium pseudotuberculosis in naturally infected sheep with caseous lymphadenitis.

Constituents of Corynebacterium pseudotuberculosis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of sonicated whole bacterial cells and ether-extracted cells revealed more than 35 bands in silver-stained gels. SDS-PAGE analysis of concentrated culture filtrates with exotoxin activity demonstrated more than 15 bands. Sera from sheep with C. pseudotuberculosis-induced disease of variable severity were used to probe immunoblots of electrophoresed ether-extracted cells and culture filtrates. Twenty or more corynebacterial molecules, ranging in molecular weight from 20 to 112 kDa, in ether-extracted cells were recognized by antibodies in the sera of naturally exposed sheep with positive ELISA titers. These sera also recognized up to six molecules, ranging from, 20 to 68.1 kDa, on immunoblots of ammonium sulfate-concentrated culture filtrate. There was no apparent relationship between the stage of disease and the response to specific corynebacterial antigens in these animals.     

Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera.

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34-38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera.     

Secreted antigens as virulence-associated markers in Staphylococcus aureus strains from rabbits

Western blot analysis was performed from the culture supernatant of 59 rabbit Staphylococcus aureus strains, classified as high and low virulence strains according to their epidemiological behaviour in commercial rabbitries, bio-, phage- and RAPD-type. Fourteen extracellular antigen bands (A-N) were recognised using sera of rabbits immunised with washed, viable high virulence S. aureus bacteria. Eleven of these bands were found in high virulence as well as in low virulence strains. The band A, approximately 78 kDa, was not seen in any of the 27 high virulence strains, except for one strain which was also typical in other aspects, was detected in all, but one of the low virulence strains. The M and N bands with molecular masses of approximately 29 and 27 kDa, respectively, were recognised in all high virulence strains except for the atypical strain, but in none of the low virulence strains. This indicates that the latter two antigens may be virulence-associated markers for S. aureus strains from rabbits.     

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.     

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin.

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87-116 kDa, 38-44 kDa and 26-28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87-116 kDa and 38-44 kDa.     

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.     

Characterization of putative Haemophilus somnus isolates from tonsils of American bison (Bison bison).

Three Haemophilus somnus isolates (2a, 3a, and 27b) and one H. somnus-like (13b) isolate from tonsils of commercially reared American bison were compared with 2 known H. somnus isolates from cattle, namely, 2336, shown to cause respiratory disease, and 129Pt, from the prepuce of an asymptomatic bull. All H. somnus isolates, but not the H. somnus-like isolate, required CO2 for growth. Biochemical utilization profiles were identical for bison and bovine H. somnus isolates with the exception of alpha-fucosidase production by isolate 3a. Isolate 27a varied from 2a, 2336 and 129Pt by hemolysis of bovine erythrocytes. Isolate 13b hemolyzed sheep but not bovine or bison erythrocytes and varied from other isolates in biochemical utilization tests. Outer membrane protein profiles of 2a, 3a and 27a were almost identical with those of bovine isolate 2336 and similar to that of 129Pt, but quite different from that of 13b. Western blots of bison isolates were similar to that of the virulent bovine 2336 isolate, including detection of high molecular mass antigens above 100 kDa and the 76 kDa antigens associated with bovine IgG2 Fc binding characteristic of virulent strains, as well as antigens of approximately 78, 60 and 40 kDa. Producers and veterinarians should be aware that H. somnus may be carried by bison and may have potential for causing diseases in bison similar to those described in cattle and sheep.     

Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis

Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.     

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium''s structure and antigenicity when cultured in vivo is discussed.     

A comparative study of bovine and ovine Haemophilus somnus isolates.

Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.     

Bovine digital dermatitis and severe virulent ovine foot rot: a common spirochaetal pathogenesis

A potential pathological role for spirochaetes in bovine digital dermatitis (bovine DD) and severe virulent ovine foot rot (SVOFR) has been considered and a treponeme isolate obtained from each disease in the UK. In this work, we have investigated the hypothesis that the two diseases may have a shared (common) spirochaetal aetiology. Experiments were designed to identify serological similarities and differences between the two spirochaetes; an enzyme-linked immunosorbent assay (ELISA) was developed to detect anti-treponeme antibodies in the sera of cows and sheep against the two-treponeme isolates. Sera were further tested for antigen reactivity by Western blotting. Cattle and sheep with bovine DD and SVOFR, respectively, had increased seropositivity rates to both treponeme isolates, with different patterns of reactivity between farms. In some cattle herds, significant correlations were shown between antibodies to bovine DD treponemes and SVOFR treponemes (P<0.001). In other herds, there was no apparent cross reaction, suggesting the presence of more than one treponeme in bovine DD on some farms. There was no significant correlation between the two treponeme isolates when ELISA-tested against 58 sheep sera from SVOFR cases (P>0.05); sheep showed strong evidence of reactivity to one or the other treponeme antigens, but never to both. Western blotting against both treponeme antigens showed that they frequently displayed different antigen epitopes, although some minor bands were common to both organisms. The data suggest that there are a number of spirochaetes in UK farms, which could be involved in the pathogenesis of either bovine DD or SVOFR.     

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides SC membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.     

Identification and characterization of corpuscular, soluble and secreted antigens of a Venezuelan isolate of Anaplasma marginale

Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax)

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of ‘incomplete’ strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the ‘complete’ M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.     

Antigen specificity and activity of ovine antibodies induced by immunization with Corynebacterium pseudotuberculosis culture filtrate

Sheep were immunized three times with a vaccine composed of filtrate from a 36 h culture of Corynebacterium pseudotuberculosis and a block polymer adjuvant. Immunization resulted in the development of exotoxin-neutralizing antibody. This corresponded to the recognition of a 31.6 kDa protein on sequential immunoblots of ammonium sulfate-precipitated filtrate. In addition sera from vaccinated sheep recognized at least eight bacterial cellular antigens on immunoblots of ether-extracted C. pseudotuberculosis, including bands of 12, 25.1, 31.6, 36.3, 39.8, 63.1, 70, 75 or 79.4 kDa. Sera from these sheep altered the colony growth characteristics of C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis antigen preparations that have been used in toxoid vaccines induces antibody responses to numerous cellular antigens in addition to exotoxin and suggest that serologically mediated antibacterial effects could be an important component in the protection from disease that has been reported following immunization with C. pseudotuberculosis toxoids.     

Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle

A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes. An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting. Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B. burgdorferi and the treponemes. Significant correlations were shown between antibodies to B. burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes. In Western blotting of B. burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands. For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa. Polyclonal antibodies to Treponema denticola and T. vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen. Monoclonal antibodies to B. burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting. The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis.