Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination.

Programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region of Nepal. From 1993 to 1996 vaccination against two endemic virulent strains of Dichelobacter nodosus was tested for its potential to contribute to the eradication of footrot from the region. Only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before their annual migration to alpine pastures were eligible for inclusion. From November 1992, the treatment of cases identified during inspections included the injection of specific vaccine. Successfully treated cases migrated with their flocks but were excluded from the vaccine trial. Non-responding cases were culled. Forty combined flocks of sheep and goats (approximately 9500 animals) were used initially to compare three vaccination regimens. Eleven flocks (sheep and goats) were treated with two doses of specific vaccine (group A), nine (sheep and goats) were treated with commercial vaccine followed by specific vaccine (group B) and 10 (sheep and goats) were treated with two doses of commercial vaccine (group C) in March to April 1993 before the annual migration; 10 flocks (sheep and goats) remained unvaccinated (group D). Only sheep and goats free of signs of footrot were allowed to migrate. Nevertheless, virulent footrot recurred in many flocks three months later. However, its prevalence was significantly lower in group A than in the other three groups combined. Groups A, B and C then received the specific vaccine before their migrations in 1994 to 1996; group D remained unvaccinated. The annual programme of inspection and identification and treatment of cases continued for seven years, but the vaccinations ceased after four years. There was no recurrence of virulent footrot after November 1993. After the first season the virulent strains of D nodosus used in the specific vaccine could no longer be isolated, although antigenically distinct, benign strains of the organism persisted in cases of benign footrot.

Diagnosis of footrot in goats: application of ELISA tests for response to antigens of Dichelobacter nodosus.

Goats are an important natural host for footrot and are infected with Dichelobacter nodosus that have virulence characteristics similar to those of sheep strains. However, the humoral response of goats to D. nodosus antigens and the possibility of a serological diagnosis of footrot in goats have not been studied. With the aim of evaluating a diagnostic ELISA test, we investigated the primary immune response of goats to experimental and natural infection, the memory response in recovered animals, and the transfer and persistence of colostral antibodies in kids. Footrot stimulated the goat's immune system and, as in sheep, under-running lesions were the primary stimulus for production of anti-D. nodosus antibodies. The immune response could be detected in ELISA using either fimbrial or outer membrane protein (KSCN) antigens of D. nodosus. Antibody titres resulting from infection declined quickly after recovery and reached pre-infection levels within 3-4 months. Previously affected animals, however, mounted a memory response when injected with purified D. nodosus antigens. Antibody levels attained after anamnestic challenge were correlated with the maximum levels attained during infection, and were therefore indicative of the infection status. Anti-D. nodosus antibodies were also transferred to kids via colostrum, but these antibodies did not persist and therefore were unlikely to interfere with the diagnostic ELISA after 3 months of age. Though these ELISA tests were highly specific, their sensitivity was rather low. Therefore, they are only suitable for a herd diagnosis of footrot in goats and are dependent on the development of advanced under-running infections in a proportion of affected goats.

Pilus ELISA and an anamnestic test for the diagnosis of virulent ovine footrot and its application in a disease control program in Nepal.

The immunological memory (anamnestic) responses in sheep recovered from virulent footrot (VFR) can be aroused by subcutaneous injection of outer membrane protein (OMP) antigens of Dichelobacter nodosus. The magnitude of this response is directly correlated to the highest antibody response attained during infection and memory lasts at least a year after recovery from VFR. However, some older animals show non-specific responses to OMP antigens. In this study an evaluation of D. nodosus pilus antigen for the anamnestic diagnosis of footrot in sheep was undertaken. The results indicated that the primary and anamnestic responses to pilus were similar in character to OMP antigen but were highly specific. The sensitivity of the procedure for detection of sheep with a history of VFR was approximately 80%. A low proportion of sheep with mild lesions due to virulent strains of D. nodosus reacted to anamnestic challenge. Anamnestic challenge with 10 microg pilus was used in a VFR surveillance program in migratory sheep flocks in Nepal. Conventional diagnostic methods could not be applied during the disease transmission periods in these flocks because of their migration to alpine pastures far away from human habitation. The results supported clinical and bacteriological findings suggesting that virulent strains of D. nodosus have apparently been eliminated from these flocks in Nepal.

Transmission of virulent footrot between sheep and goats.

OBJECTIVE: To determine the infectivity of ovine and caprine strains of Dichelobacter nodosus for both sheep and goats. DESIGN: Pen experiments in which 20 sheep and 19 goats were challenged directly with the two strains, and transmission experiments on pasture, using donors infected by experimental challenge. RESULTS: Sheep and goat strains of D nodosus infected both animal species in experimental challenges. Animals so infected transmitted footrot to both sheep and goats on pasture plots. A significantly smaller proportion of goats than sheep was infected when challenged with either strain. The interval between exposure and development of footrot in goats was longer than in sheep when recipient animals were exposed to infected donors on pasture. The disease was less invasive in goats than in sheep. CONCLUSIONS: With the strains of D nodosus used there was no evidence of host specificity. Direct transmission of footrot can occur between sheep and goats in the same environment. There is a need to include goats in ovine footrot eradication programs and vice versa.

PCR-RFLP of outer membrane proteins gene of Dichelobacter nodosus: a new tool in the epidemiology of footrot.

Currently only phenotypic epidemiological markers, serogrouping and virulence testing of Dichelobacter nodosus, are available for investigating footrot outbreaks in small ruminants. These methods have limitations in tracing the source of infection. In this study, a genotypic marker, PCR-RFLP of outer membrane protein gene, was used to characterize D. nodosus. The technique was evaluated in a controlled experiment involving two strains of bacteria. PCR-RFLP was found to be highly specific in differentiating isolates obtained from recipient animals infected with different strains. Subsequently, this technique was used to characterize isolates obtained from field cases of footrot in Nepal. A total of 11 patterns was recognized among 66 Nepalese D. nodosus isolates representing four different serogroups. PCR-RFLP also discriminated isolates with similar phenotypic characteristics. However, all isolates which, phenotypically, were virulent were represented by only two patterns irrespective of their serogroups. It is suggested that PCR-RFLP described here could be a useful epidemiological marker in the study of footrot.

Identification and characterisation of serogroup M among Nepalese isolates of Dichelobacter nodosus, the transmitting agent of footrot in small ruminants.

One thousand and sixty three isolates of Dichelobacter nodosus cultured between 1992 and 1996 from cases of footrot in sheep and goats of migratory flocks of Nepal were characterised by agglutination test using prototype antisera of the Australian classification system. Of those, sixty six isolates could not be classified into any of the nine serogroups (A-I). This study was therefore undertaken to characterise these isolates. It was established that they were agglutinated by antiserum against serotype M of an alternative classification system. The distinct antigenic character of these isolates was further confirmed by DNA sequence analysis of the gene for the fimbrial subunit protein of two of them. At a molecular level, these isolates were closer to the prototype of serogroup F, VCS 1017. However, when compared with VCS 1017, the number of amino acid substitutions (28) in the fimbrial protein of these isolates was similar to that expected between isolates of different serogroups. Because these isolates are antigenically similar to 'serotype' M, but meet all the criteria to be classified into an independent serogroup, it is proposed that these isolates together with isolates previously classified as serotype M be classified as 'serogroup M'.

Serological and virological studies of Japanese encephalitis in the Terai region of Nepal.

In 1987 and 1990, serum samples were collected from people living in the two districts (Itahari and Chitwan) of the Terai region of Nepal. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by the hemagglutination inhibition (HI) and neutralization (N) tests. By the HI test, 26 out of 172 (15.1%) sera from Chitwan and 15 out of 137 (10.9%) sera from Itahari showed positive titers. Higher positive rates were shown by the N test, where 46 out of 172 (26.7%) sera from Chitwan and 22 out of 137 (16.1%) sera from Itahari had antibodies against JE virus. A JE strain was isolated from a blood specimen of a pig raised in Kathmandu. When the nucleotide sequence of the pre-M region of the strain was compared to the same region of the other JE virus strains reported, the highest similarity was observed to the strains isolated in Nepal in 1985. These results suggest that the Terai region has been an epidemic area of JE.