Tuberculosis in free-ranging and captive wild animals: Pathological and molecular diagnosis with histomorphological differentiation of granulomatous lesions.

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface.

Diagnosis of mixed gastrointestinal nematode infection in goat by an indirect-ELISA.

An indirect-ELISA for the diagnosis of mixed gastrointestinal (GI) nematode infection comprising Oesophagostomum, Haemonchus and Trichuris species was standardized using crude somatic antigen of Oesophagostomum columbianum (CSAg-Oc) and sera of slaughtered goats with known parasitological status including Oesophagostomum, Haemonchus, and Trichuris (strong positive), Haemonchus and Trichuris (weak positive) and parasite free goats (negative). Two cut-off points, i.e. higher and lower cut-off were determined using the strong positive, weak positive and the negative control sera of goats. Thus the test sera having optical density (OD) values greater than the higher cut-off were considered positive for mixed infection with all the three nematode species, intermediate between the higher and the lower cut-off values were considered positive for mixed infection of Haemonchus and Trichuris, and less than the lower cut-off value were considered negative for any of these three nematode species. The sensitivity, specificity and accuracy of the ELISA for diagnosis of mixed GI nematodoses were 81.25, 93.18% and 90.00%, respectively, while it was 92.86% sensitive, 75.00% specific and 91.67% accurate for the diagnosis of mixed infection with Haemonchus and Trichuris. The ELISA, so standardized, detected 27.78% sero-prevalence of Oesophagostomum plus Haemonchus and Trichuris infection and 38.89% percent of Haemonchus and Trichuris infection in the field goats. The standardized assay might be exploited as a diagnostic tool and also for sero-epidemiological study of two important GI nematodes of goats.

Prevalence of Deg Nala disease in eastern India and its reproduction in buffaloes by feeding Fusarium oxysporum infested rice straw.

OBJECTIVE: To undertake a study on prevalence of Deg Nala disease in eastern states of India and to reproduce the disease in buffaloes by the Fusarium spp., isolated from the affected region. METHODS: During this investigation, a survey was conducted covering four states of eastern region to identify the Deg Nala cases as well as to isolate and characterize the causative agent(s). An experimental study was carried out to reproduce the disease in healthy male buffaloes (2-3 years age) by randomly dividing them into five groups (four in each group). Each individual group was fed with rice straw artificially infested with either of the two representative isolates of Fusarium oxysporum (F. oxysporum) (F01, F02) or representative reference strains of Fusarium equiseti (F. equiseti) (ITCCF-2470) and Fusarium moniliforme (F. moniliforme) (ITCCF-4821) for 30 days, whereas the control group was fed with normal rice straw only. RESULTS: A total of 658 Deg Nala cases were recorded and 12 Fusarium isolates were identified from the mouldy rice straw collected from these affected areas. The characterization of the isolates revealed three species viz., F. oxysporum, F. equiseti and F. moniliforme, among which F. oxysporum was predominant. The disease was artificially reproduced in three buffaloes in F01 group and one in F02 group within 20-23 days by feeding F. oxysporum infested rice straw which resembled the clinical symptoms and gross lesions of natural Deg Nala cases. CONCLUSIONS: The field investigation and laboratory studies, including experimental production of Deg Nala disease suggest the possible involvement of mycotoxins. However, further investigations needs to be done to understand nature of the toxic factors involved in production of the Deg Nala disease.

Rapid detection of Mycobacterium bovis on its lipid profile by thin layer chromatography.

Sixteen Mycobacterium bovis (M. bovis) strains isolated from bovine tissues and one standard reference strain of M. bovis AN5 alongwith other species of mycobacteria for comparison were investigated for the presence of phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) for rapid identification of M. bovis by thin-layer chromatography (TLC). The study indicated presence of PGL with an Rf value of 0.75 in chloroform-methanol solvent in all 17 M. bovis strains. The dimycocerostate A corresponding to spot A was the major constituent among all the three spots in M. bovis strains. TLC appeared to be a promising alternative to conventional biochemical methods for identification of M. bovis taking into consideration both PGL and PDIM lipids.