Stiftung Tierärztliche Hochschule Hannover (TiHo)

Control and management of avian tuberculosis in a captive collection of wildfowl at the wildfowl and wetlands trust centre in llanelli, using an enzyme-linked immunosorbent assay as diagnostic aid

Zsivanovits, Harriet Petra

With the involvement of collections of birds like those belonging to the Wildfowl and Wetlands Trust (WWT) in recovery and reintroduction programs for endangered and threatened species, disease has become a topic of considerable importance. Many diseases, such as avian tuberculosis, are difficult to detect, have long latency periods, and have no effective treatment. Because diseases with these characteristics can easily spread undetected, they represent a significant threat to both captive and wild populations in contact with infected birds. Within captive collections of birds avian tuberculosis is notoriously difficult to eradicate due to the tenacity of Mycobacterium avium which can persist in an environment for several years. It is the insidious nature and slow progress of the disease, coupled with the ability to survive outside the host, and therefore create an infectious reservoir, that makes avian tuberculosis such a problem. Therefore it is important to consider different techniques of diagnosis and to develop a management plan to control the disease. This epizootiological study is undertaken in the WWT centre at Llanelli, South Wales, where the adult mortality of avian tuberculosis rose from 20% to 43% in the last six years (1994 to 1989). First, an enzyme-linked immunosorbent assay (ELISA) was evaluated by testing blood samples from birds which were known to be positive or negative for avian tuberculosis. This ELISA was first developed by CROMIE (1991). The ELISA used in this study was now modified by using a commercially available rabbit anti-duck horseradish peroxidase conjugate and five antigens which were isolated from birds that died of avian tuberculosis in the WWT centres at Llanelli and Slimbridge. Samples which have an absorbency value above the cut-off point were considered positive and those below the cut-off point were considered negative. The results are categorises as follows: Avian tuberculosis negative: values < 20% of the adjusted positive control ; Borderline: values of 20-50% of the adjusted positive control ; Avian tuberculosis positive: values > 50% of the adjusted positive control. The ELISA was performed on blood taken from 418 birds of the collection including ducks, geese and swans. 219 birds have been repeatedly tested over the period of two years. ELISA results from those birds tested in spring and autumn show significant differences with a tendency of a higher antibody titre for avian tuberculosis, in the blood samples obtained in autumn. Further studies with serial testing twice a year for at least two years are necessary to elucidate possible seasonal changes in antibody levels. The analysis of the ELISA results to evaluate sensitivity and specificity is based on gross post mortem examination. To confirm diagnosis a histopathological examination was performed on liver and spleen biopsies of 19 birds. There was only little agreement between gross post mortem and histopathological examination with 27.8% (n=5) of questionable results and no false negative results. Therefore, the evaluation of the ELISA based only on post mortem findings as gold standard has to be considered very critically. PCR performed on 13 of the specimens prepared for histopathology did not identify the presence of mycobacterial DNA in any of the samples, therefore, confirming histopathological findings rather than gross post mortem findings. Further research, for example, identifying species-specific mycobacterial DNA fragments by molecular biological techniques such as PCR, is necessary to obtain a final diagnosis. In performing PCR the greater degree of degradation in DNA prepared from paraffin-embedded tissue compared to frozen tissue should be considered. Analysing the ELISA results by referring to such highly precise diagnostic techniques may reveal a somewhat different sensitivity and specificity than the ones obtained in the current study. 51 of the birds tested died during the period of this study and a gross post mortem examination was performed. Only the ELISA carried out in spring proved reliable with a sensitivity of 76.9% and a specificity of 55.6%. Those figures are not ideal and the percentages of false negative (23.1%) and particularly false positive results (44.4%) are not acceptable in a screening program for larger collections. Screening of individual pet birds requires an even more specific test. Some possible tests are mentioned in the literature review. Analysis of the ELISA and post mortem results according to different taxonomic tribes showed no significant differences. The ELISA results in the Dendrocygnini, Anserini, Tadornini, Somateriini and Oxyurini have to be interpreted very carefully due to a high percentage of false positive and false negative results. There is need for further reappraisal and refinement of the ELISA to improve its sensitivity and specificity as a mean of ante-mortem diagnosis of avian tuberculosis. There must also be further considerations about the evaluation of the ELISA, as basing the data on gross post mortem examination may not be accurate enough, but more specific molecular biological techniques might be advisable. Secondly, post mortem data from the last ten years were analysed to identify the distribution, prevalence and percentage mortality of avian tuberculosis in the WWT centre at Llanelli according to different sex, seasons, tribes, age and pens. The statistically significant highest mortality of avian tuberculosis was found in the North American pen, the Asian pen and the Smew pen. As possible reasons environmental details, particularly the water course through the grounds, contact to other birds of the collection and to wild birds and the former use of the grounds as farmland are discussed. The Mergini, Cairinini and Aythynini showed the highest mortality of avian tuberculosis including the very susceptible species Hooded Merganser, Smew and White-winged Wood Duck. Habitat, feeding and breeding habit and particularly genetic susceptibility are suggested to explain the different mortalities of avian tuberculosis in the different tribes. Furthermore environmental factors and the exposure to stress are considered to influence the development of disease. There was no sex predilection or differences in the occurrence of deaths due to avian tuberculosis in different seasons. There was a significant increase in the number of birds dying from avian tuberculosis over the last few years. Most notably there was a dramatic increase in mortality from 1997 (24.4%) to 1998 (40.7%), followed by a further, smaller increase in 1999 (43.4%). The increase mortality was found both in the overall number of birds and in a break down of the data according to different tribes. Finally, a management plan was proposed to support efforts to control avian tuberculosis, including general considerations and aspects specific for the WWT centre at Llanelli. These suggestions involve disinfection, sanitation, conversion programs of the enclosures with particular emphasis on the water course, husbandry of very susceptible species, avoiding contact with other birds, reducing stress, record keeping and most importantly quarantine and regular screening programs. These programmes should consist of clinical examinations and performing of an ELISA.

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Zsivanovits, Harriet Petra: Control and management of avian tuberculosis in a captive collection of wildfowl at the wildfowl and wetlands trust centre in llanelli, using an enzyme-linked immunosorbent assay as diagnostic aid. Hannover 2001. Tierärztliche Hochschule.

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