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Gross lesions were most prevalent in the jejunal lymph node, being found in nearly half of the infected animals. No other site of gross lesions approaches the same frequency of occurrence, the nearest being the retropharyngeal lymph node with a prevalence of 0.21 . This high frequency of jejunal lesions has been found in previous reports of the disease in ferrets in New Zealand (Ragg et aI., 1 995c), and provides strong evidence for the bulk of the infections having an alimentary tract ongm.

Nearly one third of the infected ferrets had no gross lesions. This satisfactorily quantifies the poor sensitivity of identification of infected animals solely by necropsy procedures. Many of the "early" lesions seen were difficult to distinguish from the variable appearance of the normal lymph node and necrotic lesions were often so small as to be barely detectable by eye.

The higher prevalence of disease found in the current series of ferrets is at odds with earlier New Zealand studies (Ragg et al., 1 995b; Walker et al. , 1 993; Cowan, 1 994; Oliver, 1 996), with the lower confidence limit in this study (0.25), being equal to the upper confidence limit of the highest previously reported. This circumstance has almost certainly arisen from inapparent infections being overlooked at necropsy, as all previous diagnoses were based on gross lesion identification at necropsy, with subsequent confirmation by histopathological or mycobacteriological examination. Some small and less advanced lesions will have been overlooked, and consequently many more AFB found in the advanced lesions that were examined. This, and earlier reports in the literature, has led to the belief that ferret lesions are characterised by enormous numbers of AFB (Symmers et at., 1 953; de Lisle et al. , 1 993). A strong relationship between the abundance of AFB and the presence of necrosis has been demonstrated in this study, which also lends weight to the suggestion that "early" lesions with few AFB have been overlooked in the past. The negative results of the poisson regression analysis are probably of most importance to understanding the disease process. The severity of disease was not related to age nor sex, indicating that disease progression is independent of these host factors. The presence of "lipid plaques", especially large ones, has a role in alerting the examiner to an increased possibility of infection, but splenic enlargement is clearly not specific enough in its aetiology to be diagnostically

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useful at necropsy. Low body condition may be associated with advanced disease, but in these cases there will be sufficient easily recognised lesions to guide the diagnosis. Despite intensive investigation there appears to be no substitute for the careful examination of lymph nodes for the presence of gross lesions, especially necrotic foci, to demonstrate the presence of infection at necropsy. Attention will be most usefully directed at the nodes with the highest prevalence of lesions in "early" cases (Table 3-II), and should include the retropharyngeal, mandibular, superficial axillary, caudal cervical, popliteal, inguinal, jejunal and colonic lymph nodes.

The data arising from the ANCOV A of body weight (Table 3-VI) suggested that male ferrets are prone to weight loss, especially in the winter when food is scarce and they are involved in mating and territorial activities, but also in the summer.

Males also showed significant weight loss when affected by advanced disease, whereas the females were not significantly affected. It would seem that males are more prone to weight fluctuation from whatever cause, than are females. These findings must be interpreted cautiously, as only 24 ferrets with advanced disease

existed in the data set, and because there is little available information on the duration of the disease process from infection to death. It seems likely that being tuberculous does not affect the general health of the ferret until the latter stages of disease. The ferrets with poorest condition in the study of Walker et al. ( 1993) had a higher prevalence of tuberculosis, which supports the notion of advanced disease affecting the health of many animals. Experimental infections have resulted in the ferrets appearing normal until a matter of days before death (Symmers, et al. , 1 953), indicating a fulmination of the disease process in the terminal stages. Our data contained one dead and one sick tuberculous ferret which were in poor condition at death or shortly prior, suggesting that the terminal stage disease may progress slowly enough for body reserve depletion in at least some ferrets. One other ferret which appeared to have died from tuberculosis still had fat reserves remaining. In possums, body condition is maintained until the terminal stage of the disease (Jackson et ai. , 1 995a).

The splenomegaly appears to be an inconsistent finding in advanced disease states, and seems to be due to an increase in mass of the normal cellular elements, and the development of small granulomas in some cases. On average the magnitude of this

enlargement IS only slight, and the effect is likely to be obscured by other

unidentified causes of hypertrophy. A large spleen found during necropsy may be associated with advanced disease, but more specific lesions will also be evident. Typical splenic lesions have been simply described as aggregations of macrophages, up to 2.5 mm in diameter, appearing as ill-defined nodules (Symmers et at. , 1 953;

Thoms et at 1 982). This is consistent with our observations. Symmers e t al. (1953) did note an enormously enlarged spleen in one experimentally infected ferret. Splenomegaly is a consistent feature of the disease in mink, in which the disease process generally seems to be more severe (Pulling, 1 952; Head, 1 959; Adamesteanu et al. , 1 970; Beck et aI., 1 974).

No gross hepatic lesions were evident in the current series of ferrets, but these have been reported in one of 98 infected ferrets by Ragg et al. ( 1995c), and by Dunkin et al. ( 1929) in two naturally infected animals. Despite the lack of gross lesions, microscopic liver granulomas were the most prevalent lesion of infected ferrets and have been reported in all other cases where the liver has been examined. These granulomas may, however, be absent in early infections or in cases where the infection has been localised to a peripheral lymph node. Because of the very high specificity of these lesions we believe that they are pathognomonic for M bovis infection, even in the absence of visible AFB. The high prevalence of these granulomas will make rapid diagnosis by histology a possibility for cross-sectional studies. The diagnostic potential of liver biopsy may have some experimental utility, and could be used as a tool in longitudinal studies. Sensitivity could be improved by the use of a wider bore biopsy needle, allowing the collection of a larger volume of liver. As all these granulomas will undoubtedly contain some AFB, sections of liver may be usefully incorporated into pooled tissue samples for mycobacteriology, or polymerase chain reaction (peR) testing if a rapid definitive diagnosis is required.

None of the ferrets we examined had extensive nor advanced pulmonary lesions. The common occurrence of other forms of granulomatous lung disease means that histological examination of lungs for tuberculous lesions will not be diagnostic. Although one case of a caseating pulmonary lesion has been reported (Dunkin et al. , 1929), necrosis of pulmonary granulomas, even those containing AFB, was not present in the current series. The absence of necrosis and extensive lesions

indicates that the cell-mediated immunity of the pulmonary compartment, unlike many species, can provide an environment which is hostile to the survival of M bovis, and also that primary infection of the lungs is rare. Ragg et ai.(1995c) also found a low prevalence of pulmonary lesions (2.9%) attributable to tuberculosis. Symmers et ai. ( 1 953) believed that the entrapment of AFB in "lipid plaques", which are common in the genus Mustela, was secondary to their formation from unknown causes. We believe this to be true, but our findings also suggest that the presence of M. bovis in the pulmonary circulation may exacerbate pre-existing lesions and make visible those that would have hitherto been inapparent.

The results of the log-linear analysis allowed further strengthening of hypotheses regarding the pathogenesis of infection. The association between lesions in the head area and the caudal cervical lymph node is readily explained by the efferent lymphatic branch of the retropharyngeal node which passes to the caudal cervical (Shibata, et ai. , 1 988). Head lesions were also statistically associated with gut­ associated lesions, which is consistent with the alimentary tract, including oral mucosal injuries, oropharyngeal tonsils and intestinal Peyer's patches, being the principal route of entry of bacilli when ingested. The association of head lesions with thoracic lesions is probably similar to the relationship between the peripheral nodes and the thorax, in that bacilli entering the circulation from these sites must first pass through the pulmonary capillary bed where they are likely to be engulfed by intravascular or intra-Iesional macrophages. The association between the HAEM group of sites and thoracic lesions provides strong evidence that when spleen, bone marrow, kidney, and adrenal gland become involved, which can only be through haematogenous spread, the lung is also likely to be infected simultaneously. The almost universal infection of the liver, no matter what other sites were infected, demonstrates that bacilli readily escape from nodal lesions, and are distributed by the blood stream early in the course of infection and are trapped by the Kupfer cells lining the liver sinusoids.

It appears that the disease process for M. bovis infection in the ferret is not as aggressive and tissue destructive as it is in many other species in which the lesions are normally caseous in nature. The histological appearance of advanced lesions in the genus Musteia is characterised by macrophage accumulation, large numbers of

containing multiple bacilli. Giant cells are not a feature, and caseation and fibrosis

rare (Dunkin et al. 1 929; Head, 1 959; Adamesteanu et a!., 1 970; Thorns et a!.,

1 982). Large numbers of M. bovis organisms have been found to be toxic for ferret

lymphocytes in vitro (Thorns et aI. , 1 982), and also apparently for macrophages in

vivo. The development of the cell-mediated response appears to be slow, and is

possibly compromised by the presence of M bovis antigens, which are capable of

suppressing lymphocyte reactivity (Thoms et a!., 1982). Ferrets, once infected,

keep the disease under control for an extended period of time, during which the

lesions progress slowly, until sufficient M. bovis and their secreted antigens are

present to cause a very severe depression of the cell-mediated immune response. The organism then replicates freely and death ensues, presumably from toxaemia. Ferrets, because of their high prevalence of infection and widespread distribution, are potentially a very good indicator of wildlife infection in an area. As the capture yield is low it is worthwhile ensuring that those which are caught will be accurately classified with regard to tuberculosis status. Because many tuberculous ferrets will have inapparent infection at necropsy, it is essential that additional laboratory procedures are carried out on animals without lesions. Examination of the liver of all ferrets by histopathology is strongly recommended as a rapid and sensitive diagnostic test. Where tissues are being sent for culture in the absence of gross lesions, a pool of retropharyngeal, caudal cervical, jejunal lymph nodes and a small portion of liver should be collected for mycobacteriological examination, to ensure the greatest likelihood of including sufficient bacilli for successful isolation from a single pooled sample.