Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-09T19:52:40Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CAROLINE DA SILVA SILVEIRA.pdf: 6734641 bytes, checksum: 8df9bfda7306dbc3b2714620b6612002 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-09T19:52:54Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CAROLINE DA SILVA SILVEIRA.pdf: 6734641 bytes, checksum: 8df9bfda7306dbc3b2714620b6612002 (MD5) / Made available in DSpace on 2016-09-09T19:52:54Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CAROLINE DA SILVA SILVEIRA.pdf: 6734641 bytes, checksum: 8df9bfda7306dbc3b2714620b6612002 (MD5) Previous issue date: 2016-02-23 / Doenças podais são uma das principais injúrias em rebanhos de pequenos ruminantes em diversos países e a pododermatite infecciosa (Footrot) é relatada como a mais frequente em ovinos no Brasil. No Rio Grande do Sul, as doenças podais ainda são um grave problema para os criadores de ovinos e pouco tem sido feito para saná-las. O Footrot, mesmo se tratando de uma doença de notificação obrigatória e frequente na região, os registros oficiais sobre a situação da doença nos rebanhos são escassos. Esse trabalho teve como objetivo descrever as principais características das lesões podais observadas em ovinos da Mesorregião Sudoeste do Rio Grande do Sul, com ênfase nos aspectos epidemiológicos, macroscópicos, microscópicos e radiográficos das lesões de Footrot. O estudo foi realizado em duas etapas. Inicialmente foram avaliados ovinos em 27 propriedades rurais, das quais 21 registraram a ocorrência de lesões podais em ovinos com perdas econômicas significativas. Aproximadamente 1.700 ovinos, em média 10% dos animais do rebanho, apresentavam diferentes graus de claudicação decorrente de lesões podais que, macroscopicamente, variavam de brandas a severas. Posteriormente, foram avaliados os variados graus de lesões de Footrot nos ovinos. Em casos de abate e necropsia, os cascos dos ovinos com as lesões foram submetidos à avaliação macroscópica, radiográfica e microscópica. Dessa forma a doença foi classificada em cinco graus de severidade que variaram de 1 (lesões leves) a 5 (lesões graves). Verificou-se que diversos fatores como clima e manejo foram favoráveis para o desenvolvimento das lesões podais e essas estão associadas, na maioria dos casos, a Footrot em diferentes estágios de evolução. A partir dessa classificação em graus foi possível classificá-los macroscopicamente em duas síndromes clínicas propostas, a saber Footrot benigno e maligno. Essa classificação facilita o estabelecimento das medidas de controle com intuito de limitar a propagação da doença e evitar a evolução das lesões nos cascos acometidos. / Foot diseases are one of the main disorders in small ruminant flocks in several countries and infectious pododermatite (Footrot) is reported as the most frequent podal lesions in sheep in Brazil. In Rio Grande do Sul state, foot diseases still a serious problem for sheep farmers and little has been done to remedy them. Footrot is a notifiable disease and frequent in the region, the official records on the disease situation in herds are scarce. This study aimed to describe the main features of foot lesions observed in sheep from Mesoregion Southwest of Rio Grande do Sul, with emphasis on epidemiology, macroscopic, microscopic and radiographic changes of Footrot injuries. The study was conducted in two steps. Initially, sheep were evaluated on 27 farms, of which 21 showed records of the occurrence of foot lesions in sheep and significant economic losses. Approximately 1,700 sheep, about 10% of the flocks, showed varying degrees of lameness due to foot lesions, macroscopically characterized as mild to severe. Subsequently, they assessed the varying degrees of injuries Footrot in sheep. Hooves with injuries were submitted to macroscopic, radiographic and microscopic evaluation. Thus the disease was classified into five grades of severity ranging from 1 (mild injury) to 5 (severe damage). It has been found that several factors such as weather and handling were favorable for the development of foot injuries and these are associated in most Footrot cases in different stages of evolution. Based on the classification in degrees of infectious pododermatitis it was possible to classify them macroscopically in clinical syndromes proposed as benign and malignant Footrot. This classification facilitates the establishment of control measures with the intention of reduce spread of disease and prevent the development of lesions in affected hooves.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

Footrot is a contagious hoof disease of ruminants. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. The disease results from a mixed bacterial infection, but the essential agent is Dichelobacter nodosus, a Gram-negative, anaerobic bacterium that possesses type-IV fimbriae on its surface. Genetic variation in the fimbriae of D. nodosus was investigated in this study. Using the polymerase chain reaction (PCR), the variable region of the gene encoding the fimbrial subunit (fimA) was amplified from bacterial DNA extracted from footrot lesions. Different fimA amplimers were differentiated by single-strand conformation polymorphism (SSCP) analysis. In conjunction with DNA sequencing, 15 unique sequences of D. nodosus fimA were obtained from 14 footrot samples taken from 6 farming regions throughout New Zealand. When these sequences were compared to fimA of known serogroups, it revealed that there were at least 15 D. nodosus strains, representing 8 serogroups, present on New Zealand farms. The predominant serogroup was B which contained 6 strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Twelve out of the 15 New Zealand D. nodosus strains had fimbriae different to those previously reported and the presence of multiple strains on a single hoof was common (86% of samples). The fimA sequences from the 12 D. nodosus strains incorporated into the footrot vaccine currently available in New Zealand were determined. A primer set targeting the relatively conserved fimA regions and based on the published sequence of serogroup M Nepalese isolates (designated M-Nep), failed to amplify fimA from the vaccine serotype M strain (designated as M-SPAHL). When the downstream primer was substituted with a primer that was specific for other serogroups of D. nodosus, the fimA gene was successfully amplified. Cloning followed by DNA sequencing, revealed that M-SPAHL fimA was different to M-Nep fimA. The predicted amino acid sequence of M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not M-Nep. The sequence difference between M-SPAHL and M-Nep was larger than that expected within a serogroup. The consequences of serological relatedness and sequence dissimilarity are discussed. Only eight of the 15 New Zealand field strains had fimbriae identical to those of the vaccine strains, while the remaining seven strains possessed different fimbriae. In addition, the vaccine contained two more D. nodosus strains, representing two sera groups, that were not found on the New Zealand farms investigated in this study. This may, to some extent, explain why the current footrot vaccine is at times less efficient in New Zealand. Another 17 footrot samples were screened for new or additional D. nodosus strains. Two PCR amplimers (designated X and Y) derived from footrot samples generated SSCP patterns different to those of previously identified strains. DNA sequencing revealed that these two fragments possessed novel sequences. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1 whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M1 or E1, produced PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M1 and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artefacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It therefore seems more likely that fragments X and Y reflect genuine fimA sequences of D. nodosus which have resulted from in vivo DNA recombination, than from a PCR recombination artifact. The genetic capability for recombination at the fimbrial subunit locus may therefore endow D. nodosus with the ability to alter its antigenic appearance. D. nodosus strains present in footrot lesions can be genotyped using a PCR-SSCP/sequencing technique. However, this typing technique requires cloning and screening of D. nodosus fimA sequences, which is both laborious and costly. A rapid molecular typing system for D. nodosus was therefore developed in this study. A close examination of available D. nodosus fimA sequences revealed regions that appear to be specific for serogroups and serotypes. These regions were used to design a panel of sequence-specific oligonucleotide probes (SSOPs), and a rapid and accurate D. nodosus typing system using PCR and reverse dot-blot hybridisation (PCR/oligotyping) was subsequently developed. The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific, poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments that had hybridised were detected. The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing, was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types were present in the original footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus present in a footrot sample can be accurately genotyped in less than two days. Individual animals from the same farm, or the same paddock, were often infected by different strains of D. nodosus. This suggests a host role in mediating footrot infection, or that the interaction between the pathogen and the host is important. In order to better understand the interaction between the bacterium and the host, two polymorphic ovine class II MHC genes DQA1 and DQA2, which have been previously shown to be important in footrot infection, were also investigated in this study. PCR-SSCP/sequencing analysis of the DQA1 locus revealed ten unique ovine DQA1 sequences, with five of them being newly identified. This increases the number of known ovine DQA1 alleles from 8 to 13 (including a null allele), implying a high level of polymorphism at the ovine DQA1 locus. D. nodosus present on 20 footrot infected sheep from the same flock were genotyped, together with the ovine DQA1 and DQA2 genotypes of their hosts. Preliminary results showed that sheep with the same DQA1 and DQA2 genotypes tended to be infected by similar types of D. nodosus. Different types of D. nodosus were generally found on sheep with different genotypes at either the DQA1 or the DQA2 locus. This suggests the diversity in D. nodosus infection may be associated with the heterogeneity in the host MHC. However, as only a small number of animals from the same sire were analysed, further investigation is needed to gain a better understanding of the interaction between D. nodosus and the host MHC.