Introduction

Paratuberculosis or Johne’s disease (JD) is chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis. The disease occurs worldwide and is primarily a disease of domesticated ruminants, including cattle (both beef and dairy), sheep, goats and farmed deer. This is a deadly intestinal ailment which causes weight loss, diarrhoea (intermittent or continuous) and emaciation and has significant impact on the global economy (Sweeney, 1996). Losses result from reduced productivity in terms of milk yield (quality and quantity), shorter life expectancy, reduced fertility, longer calving interval, premature culling, reduced salvage value at slaughter, increased treatment cost and risk of contracting and culling due to other diseases (McNab et al., 1991). Despite high priority, John’s disease continues to cause huge losses (200million USD per year) to US cattle industry (Ott et al., 1999). However in India JD is not a priority, despite low per animal productivity. Information on prevalence and production losses caused by JD in 465.50 million domestic ruminants has neither been estimated nor realized, mainly due to lack of diagnostic kits and reagents and control measures. Diagnosis of JD is difficult due to long incubation period, absence of characteristic symptoms, problems in cultivation of MAP and nonspecific results in Johnin test. The macroscopic and histologic lesions remain confined to the intestine, mesenteric and ileo-caecal lymph nodes (Jubb et al., 2007) hence the infected animal becomes shedder for this MAP organism in faeces. Therefore, the present study was conducted to detect the MAP infection in buffaloes by using faecal microcopy via acid fast stain examination.

Materials and Methods

The present study was conducted in Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Mhow, Indore (M.P.). A total of 150 buffaloes were screened for the present study from slaughter houses of Cantonment Board, Mhow and Nagar Nigam, Indore, belonging to different places of Malwa region, viz. Depalpur, Dewas, Mhow, Dhar (Sundrel), Indore and Maheshwar aged between 1 to 10 years, were subjected to postmortem examination. For this purpose, the slaughterhouses were visited daily for a period of eight months (September 2015 to April 2016) for collection of faecal samples. Approximately, 5-10 gm faecal sample from each buffalo before slaughter was collected in the labeled polythene bags from the rectum along with rectal pinch and collected faecal samples were transported to the Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Mhow, and kept at -20^oC in deep freeze for detection of Mycobacterium avium subsp. paratuberculosis (MAP) organism.

Processing of Faecal Samples (For Microscopic Examination)

Approximately, 2 g of faecal sample was finely triturated in the sterilized pestle and mortar with the help of sterilized distilled water (10-12 ml). Triturated material was transferred to 15 ml centrifuge tubes. After that tubes were centrifuged at 1557 X g for 1 hr at room temperature. Supernatant was discarded and middle layer was decontaminated in 25 ml of 0.9% Hexadecyl Pyridinium Chloride (HPC) for 18-24 hrs at room temperature. After decontamination and sedimentation, the supernatant was removed slowly and of about 1 ml of sediment left in the test tube was used for preparation of smears with the help of wooden stick on clean and grease free slides. After making the smear it was air dried.

Staining of Faecal Smear by Ziehl – Neelsen Staining Method

Smears were prepared by using residual faecal sediment on clean and grease free slides were flooded with carbol fuschin for 10 minutes while exposed to a steam bath to allow maximum penetration of dye in to the bacterial cell wall. Then the slides were cooled to the room temperature and then rinsed thoroughly with tap water. After that the slides were rinsed with a continuous stream of acid alcohol reagent for 30 seconds followed by distilled water wash. Methylene blue was used as a counter stain to cover the slides for 1 minute before final distilled water rinse. Slides displaying pink coloured short rods (0.5–1.5 µm), indistinguishable to Mycobacterium avium subsp. paratuberculosis (MAP) was considered as positive for Johne’s disease/MAP infection in the faecal smear examination.

Results and Discussion

In the present study, 150 (120 females and 30 males) buffaloes slaughtered at Cantonment Board slaughter house, Mhow and Nagar Nigam, Indore were examined for Mycobacterium avium  subsp. paratuberculosis in the faecal smears, prepared after processing the faecal samples for microscopic examination in 0.09% HPC, and stained by Ziehl- Neelsen’s (Acid fast staining) method. Based on the presence of pink coloured short rod (0.5 – 1.5 µm) shaped MAP bacteria by fecal smear examination revealed 79.33% (119/150) overall incidence of MAP infection in buffaloes of Malwa region of Madhya Pradesh (Plate 1 and Table 1).

Table 1: Incidence of acid fast bacteria in faecal smear examination

In the present study, MAP organisms were detected by using faecal microscopy in apparently healthy buffaloes aged between 1 to 10 year with no history of clinical John’s disease. Direct microscopy was used for the examination of clinical samples (faeces, blood, milk, tissue and necropsy samples) for detection of MAP. Ziehl-Neelsen (ZN) or acid fast based microscopy has the advantage of being simple, fast and inexpensive.

The faeces being a major source of infection for disease transmission and hence considered the first choice as a clinical specimen, though blood, milk and tissue samples are equally good for detection of MAP. A presumptive identification of pTB (Paratuberculosis) can be made if clumps of acid fast bacilli are seen under the microscope in ZN stained smears (Chaubey et al., 2016). Accordingly, the present study also recorded the incidence of MAP by screening   faecal samples of buffaloes of Malwa region (Madhya Pradesh).

In the present study, 150 faecal samples of buffaloes were examined for MAP infection after processing in 0.9% HPC and staining by Ziehl-Neelsen’s method. The overall incidence of MAP in faecal smears was found to be 79.33% (119/150) based on the presence of pink coloured MAP organisms in the faecal microscopy. The findings of the present study are in close proximity with the findings of Tripathi et al. (2006) with 72.2% infection rate in goats, Kumar et al. (2008) with 66.6% infection by direct microscopy of faecal samples of Nilgai in Mathura region, Singh et al.(2010) with 66.7% infection in Barberi kids, Kaur et al. (2011) with 46.40% infection by direct microscopy in cattle, buffaloes, goat and sheep, Singh et al. (2011) with 79.6%infection, Singh et al. (2012) with 59.9% infection in Jamunapari goats of CIRG, Makhdoom and Rawat et al. (2014) with 68.5% infection by direct microscopy in HF cows. The high incidence recorded in the current study is in contrast with the findings of Singh et al. (2007) with 38% rate of infection from Mathura, Kumar et al. (2008) with 33.3% infection in Nilgai population of Mathura region, Misra et al. (2009) with 30.85% infection in Mathura district, Shisodiya et al. (2009) with 36% infection in animal keepers, Barad et al. (2013) and Singh et al. (2013) with 28% in goats, Kumar et al. (2014) with 38% infection in faecal samples of cows and Garg et al. (2015) with 36.9% infection from dairy farms located in the Punjab state of India. The higher rate of incidence recorded in the present study is attributable to the fact that, the present survey was carried out in slaughtered buffaloes which are being culled owing to their unproductivity, as culling of unproductive buffaloes is the routine practice of dairy farmers of Malwa region of Madhya Pradesh.

Conclusion

The present study demonstrates the faecal smear examination as a rapid and quick diagnostic method for estimation of clinical and subclinical paratuberculosis in absence of molecular and histopathological techniques in field condition.

Acknowledgment

The authors acknowledge the research facilities provided by the College of Veterinary Science and Animal Husbandry, Mhow, Indore, Madhya Pradesh, India, for conducting this study.

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