Detection of Mycobacterium avium subsp. paratuberculosis (MAP) from Subclinical Caprine Paratuberculosis Cases of Odisha

Small ruminants play a vital role in the lives of poor, landless, marginal and small farmers in arid and semiarid rainfed regions of India. Goat and sheep chiefly constitute the meat-producing animals in India and is readily preferred among the people irrespective any social taboo (Kumar and Roy, 2013). The livestock wealth of the country can be well judged by its 2^nd position in goats and 3^rd position all over the world (FAO 2014). There are around 200.23 millions of small ruminants (135.17 million goats and 65.06 million sheep) in the country and reared for the production of meat (19^th Livestock Census - 2012). Further, milk, fibre and manure from them contribute to the economic value.  In meat production, India stands fifth in the world and contributes for 3% of the total world meat production of 220 million tons (Red Meat Manual, 2009). Odisha also contributes handsomely in the population of small ruminants with 4.8% of goats population (6.5 million) and 2.3% of sheep population (1.5 million) (19^th Livestock Census - 2012).
       
In spite of being a strong contributor to the income and livelihoods of poorest section of society the health status of small ruminants is neglected at the national level (Chakraborty and Gupta, 2017). The goat sector is constrained by several factors like limited or no access to treatment or diagnosis of diseases, which incurs significant economic and production losses. Among those, paratuberculosis or Johne’s disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (MAP) has emerged as a major animal pathogen (Singh et al., 2013). Johne’s disease (JD) is chronic wasting disease characterised by occasional diarrhoea, weight loss, emaciation due to protein losing enteropathy in sheep and goats (Otta et al., 1999; Manning and Collins, 2001). The disease has been reported from both large and small ruminants of India (Singh et al., 2010a).
       
But the diagnosis of JD in small ruminants is very difficult due to absence of characteristic symptoms as exhibited by large ruminants. Diagnosis is mainly dependent upon the shedding of acid-fast bacilli determined by Ziehl-Neelsen staining of faecal smears (Bhat et al., 2018). However, it is most reliable only in clinical cases (Collins et al., 1993). Cultivation of faecal and intestinal tissues (Sockett et al., 1992; Cocito et al., 1994) is the best method but is very time-consuming and less sensitive in nature. Therefore, a rapid diagnostic test IS900 PCR is developed for detection of MAP from faeces and blood which is equivalent in sensitivity to bacterial culture (Tripathi and Stevenson, 2010; Bhide et al., 2006; Singh et al., 2010b; Dhama et al., 2011). Indirect ELISA is the antibody assay used which is useful in detection of antibodies at early stage of clinical infection (Kurade, 1999; Singh et al., 2008). Though bio-prevalence of JD has been reported in different parts of country, however it has not been explored in our state. Therefore, this study is aimed to carry out a comprehensive study on bio-prevalence of MAP in 4 different districts of Odisha by using different diagnostic tests that include conventional, serological and molecular techniques.

Animals and samples
 
The present study was conducted in the Animal Health Division, Central Institute for Research on Goats (CIRG), Mathura district, Uttar Pradesh. A total of 121 goats of aged between 1 - 10 years were screened for the present study from goat farms of Odisha belonging to 4 different districts viz. Nayagarh, Cuttack, Khordha and Angul aged between 1- 10 years. Blood (n-121), serum (n-121) and faecal (n-16) were screened for MAP infection using blood PCR, ELISA and microscopic examination. Blood samples (1ml) each were collected in 2.7% EDTA and clot activator vaccutainer tube and serum was separated using centrifugation at 4500 rpm for 5 minutes. Faecal samples were collected through rectal pinch method.
 
Detection of MAP
Microscopic Examination of Ziehl Neelsen Staining Fecal Smear
 
About 2 gm of fecal sample was homogenized in 3- 4mL of sterile normal saline solution (NSS) in pestle mortar and made into a fine paste. This paste was transferred to 15mL centrifuge tubes after diluting with 7-8mL of sterile NSS. The solution was centrifuged at 3500 x g for 45 minutes to concentrate bacilli. Following centrifugation, the top layer was decanted, the semisolid middle layer was used to prepare a thin layer of smear over the glass slide. Smear was heat fixed and stained with Ziehl Neelsen’s stain and visualized under the microscope (100X) for pink coloured small rods (Singh et al., 2010b).Shedding of MAP was measured as quantitative (+1 as low shedders, +2, +3 as moderate shedders and +4 as heavy shedders) and any animal that was found positive (+1 to +4 scale) was taken as positive for MAP infection (Singh et al., 2013c).
 
ELISA Testing
 
Serum samples were screened by ‘indigenous ELISA kit’ as per the method of Singh et al., (2007). Briefly, 0.04µg per well of MAP ‘Indian Bison Type’ protoplasmic antigen was taken in 10ml of carbonate-bicarbonate buffer (pH 9.6) and coated in duplicate wells of flat bottom 96 well ELISA plates. Plates were incubated at 4°C overnight, washed thrice with washing buffer {Phosphate Buffer Saline (PBS) + 0.05% Tween-20, pH - 7.4 PBST}, blocked (3% skimmed milk powder in PBS) and incubated at 37°C for 1h, plates were washed thrice with PBST and stored at 4°C till further use. Test serum samples were adsorbed overnight at 4°C with 2 mg/mL Mycobacterium phlei (Allied Monitor Inc., USA) and then serum dilution (1:50) in 1% Bovine Serum Albumin (BSA). After incubation, 100µL of each test serum was transferred to duplicate wells of ELISA plates and plates were incubated for 2h at 37°C. Then, the plates were washed thrice with  PBST. After washing, 100µL of diluted (1:6000) rabbit anti-goat horse peroxidase conjugate was added to wells and incubated at 37°C for 1h. Plates were washed thrice with PBST and 100µL of freshly prepared substrate o-Phenylenediamine dihydrochloride (OPD, 5mg per plate at pH 5.0) was added to each well and incubated in dark for 15 min at 37°C. Reaction was stopped by adding 0.1N HCl. Absorbance was taken at 450 nm in an ELISA reader. Since JD is an insubstantial disease, rather of having a single point cutoff, OD (Absorbance) values of ELISA were converted to sample to positive ratio (S/P ratio) standardized and categorized by Collins (2002). Categories are if S/P ratio is 0.00-0.09 (Negative), 0.10-0.24 (Suspected or Borderline), 0.25-0.39 (Low positive), 0.40-0.99 (Positive), 1.00-10.0 (Strong positive) as per Collins (2002). As the disease is endemic in the country, goats in positive and strong positive categories were taken as positive for the infection.
 
Sample to Positive ratio was calculated (Collins, 2002):

 

 
Isolation of DNA and Blood PCR
 
Isolation of DNA from blood was carried out as per the experimental conditions described by Singh et al., (2010c). MAP specific primers unique to MAP (IS900 P 90/91) as per Millar et al., (1996) were procured. Primers sequences used were
 
(i) Forward primer-
P90B 5’-GAA GGG TGT TCG GGGCCGTCG CTT AGG -3’
 
(ii) Reverse primer-
P91B 5’-GGC GTT GAG GTC GATCGC CCA CGT GAC -3’
 
Briefly the reaction conditions were 94°C, 3 minutes (initial denaturation) for one cycle, 94°C, 30 seconds (denaturation), 63°C, 15 seconds (annealing), 72°C, 1 minutes (extension) for 30 cycles and a final extension of 72°C, 10 minutes for 1cycle and stored at 4°C.
 
Genotyping of MAP Infection by IS1311 PCR-REA
 
IS1311 PCR genotyping was carried out using M56 and M119 primers as per Sevilla et al., (2005). PCR products (608 bp) were digested using HinfI and Mse I enzymes and patterns were compared as ‘Cattle type’ or ‘Sheep type’ or ‘India Bison type’ as per (Whittington et al., 2001).

Detection of MAP by microscopic examination
 
Screening of 16 pooled faecal samples collected from 4 different districts of Odisha viz. Nayagarh, Cuttack, Khordha and Angul by microscopic examination revealed 62.5% (10) positive for AFB, indistinguishable for MAP (Table 1). All the 16 positive samples showed moderate shedding of AFB (+1 and +2 reactivity to ZN staining) (Fig 1).
 
Detection of Anti-MAP Antibodies by ELISA
 
Out of the 121 serum samples collected from 4 different districts of Odisha screened, 23 (19.01%), 85 (70.25%), 5 (4.13%), 8 (6.61%) were found strongly positive, positive, low positive and negative for Anti-MAP Antibodies respectively (Table 2). Among the 4 districts of Odisha, Angul registered 23 (19.01%) all the strongly positive cases of Anti-MAP Antibodies (Table 3).
 
Detection of MAP by IS900 PCR
 
Positive PCR products for MAP were detected as 413 bp product using specific IS900 PCR (Fig 2). Out of the 121 screened serum samples having positive and strongly positive categaory, 11 (9.01%) were found to be positive in ‘blood PCR’ (Table 4).
 
Genotyping of MAP
 
MAP DNA of positive goats was genotyped by IS1311 PCR-REA. Positive PCR products using specific IS1311 primers were recognised as a 608 bp product. Restriction endonuclease analysis of 608 bp PCR product developed by digesting with HinfI and MseI restriction enzymes resulted into three digested products of 323, 218 and 67 bp size, concluded an ‘Indian Bison type’ pattern similar to the positive control (Fig 3).
       
Sampling of desired animals (both clinical and subclinical cases) is found to be one of the major constrains in developing countries like India. Therefore, detail information regarding national bio-prevalence of caprine diseases and the losses caused by them is limited only to developed countries. In these type of prevailing conditions of our country, it is difficult to picturize a clear picture of chronic diseases like Johne’s Disease where clinical symptoms are not that much prominent like ruminants. This has also lead into ineffective control of paratuberculosis (Mukartal et al., 2017; Bhat et al., 2018).
       
Johne’s disease causes serious financial and productive losses to farmers rearing sheep and goat. As the disease goes unidentifiable in the early stages, still early diagnosis is very much crucial for effective control of disease in herds (Singh et al., 2010c). Earlier studies related to clinical JD with respect to its bacteriology, immunology, histopathology and their inter-relationships failed to provide much relevant information on septicaemia and its transportation in blood-stream (Van Kruiningen et al., 1986; Clarke and Little, 1996; Burrells et al., 1998). Basing upon the findings of further studies, it is likely to be assumed that MAP septicaemia and transmission occurs in subclnical stages prior to development of clinical signs (Gwozdz et al., 2000; Barrington et al., 2003; Sohal et al., 2007; Chaubey et al., 2016). Therefore, it is prudent to use multiple screening tests to detect infection at early stages.
       
This is arguably the first report on bio-prevalence of MAP infection in goats in coastal districts of Odisha. ZN staining provides information regarding shedding load and shedding pattern of AFB by the animals which is beneficial in terms of diagnosing the animal either in clinical or subclinical infectious stages. Our results shows 62.5% prevalence of MAP in pooled samples collected from farms of 4 different districts viz. Nayagarh, Cuttack, Khordha and Angul depicting sub-clinical form of disease in the state. Our results is in accordance with the findings of Bhat et al., (2018) and Shah et al., (2012) who reported 35.7% and 34% prevalence in Ganderbal district and Bandipore district of Kashmir valley respectively. Singh et al., (2013a) reported 21.6% prevalence of MAP infection in goats of eight different states through ZN staining of faecal smears. In India, a highest prevalence of MAP infection (77.5%) was reported in CIRG goats, Makhdoom (Singh et al., 2010a). Barad (2009) and Singh et al., (2013d) reported 8.93% (5 out of 56) and 28% (14 out of 50) prevalence of MAP infection respectively in goats of Gujrat. Singh et al., (2014) also reported low (32.7%) bio-load of MAP in goat population in the last 28 years (1985 to 2013) of survey in India. Our results shows 62.5% prevalence of MAP in the farms of 4 different districts viz. Nayagarh, Cuttack, Khordha and Angul. This finding lead to the conclusion that prevalence of MAP was higher in studied areas as compared to earlier reported states depicting sub-clinical form of Johne’s disease in the Odisha state.
       
Efficacy of a diagnostic test for a subclinical infection can be best evaluated through serological surveys because sub clinically infected animals represent the reservoir of a disease in a herd (Sohal et al., 2007). And ELISA is the most sensitive, specific and cost effective test to screen serum antibodies against M. paratuberculosis in a herd (OIE Manual, 2000). Our ELISA results suggest 19.01% strong positive cases and 70.25% positive cases as evidenced from 4 different farms of Odisha. Our results are in accordance with the study of Tripathi et al., (2006) who reported a higher sensitivity of 91.6% in all the clinically affected goats. Sweeny et al., (1995) also reported 87.0% sensitivity of ELISA in animals showing clinical signs of diarrhoea and wasting as compared to a low sensitivity of 15% in sub clinically affected animals. Depending upon the level of infection and high microbial loads in goat herds, sensitivities of 54-90% have been reported (Garcia Marin et al., 1991; Burnside and Rowley, 1994; Rajukumar et al., 2001). Barad et al., (2013) observed a moderate 43.3% sensitivity in Mehsana goat breed of Gujrat sub clinically affected with JD. Five positive animals in low positive category of ELISA were found negative in Blood PCR may be they are silent or low shedders having low DNA copy number unable to amplify in PCR.
       
Considering PCR as a powerful diagnostic tool to specifically amplify the DNA of MAP, a significant proportion of goats were screened using IS900 PCR on DNA (extracted from blood) to obtain the frequency of distribution of MAP in young kids and adult goats (Gwozdz et al., 1997). Out of 121 blood samples, a very low i.e. 11 (9.01%) showed positivity for MAP in 4 coastal districts of Odisha. Our facts are in accordance with the studies conducted by Singh et al., (2010c) who reported a moderate positivity (42.8%) in the blood samples collected from outside the CIRG farm as compared to a high reactivity (87.5%) in the samples collected from CIRG farm. Since CIRG herd animals come under the endemic zone for JD, therefore it shows higher prevalence of MAP. Presence of MAP in blood PCR also indicates the dissemination of organisms into intestines through RBC (Hines et al., 1987). Therefore, other studies, Munjal et al., (2005) detected IS900 sequences in mesenteric lymph nodes of 10% of 277 goats screened.
       
As the reports of similarity of IS900 sequences with other mycobacterial species (Englund et al., 2002), specificity of PCR product was confirmed by restriction enzyme (RE) and sequence analyses (Sevilla et al., 2005) in order to study the molecular epidemiology of MAP infection affecting different species of livestock residing in different geographical areas of our country. IS1311 PCR-REA revealed an “Indian Bison type” pattern in 2 samples (1.64%). “Indian Bison type” was the major biotype (97.0%) affecting goat, sheep, cattle and buffalo population of our country as evidenced by the molecular epidemiological study within the duration of 2004-2013 (Singh et al., 2014). In another study, evaluation of 80 MAP DNA samples collected from different countries 63 samples genotyped as “Bison type” and all the samples were obtained from MAP infected animals of north India. Contrary to this study “cattle type” genotype was found to be predominant in ruminants and non-ruminants of other countries (Singh et al., 2015).

MAP is an important animal pathogen of livestock industry worldwide. Bio-load of MAP is always high in small ruminants even in apparently healthy animals that were slaughtered for meat production and in animals that died due to other diseases. According to the detailed geographical zone-wise study conducted by Singh et al., (2014), present study reported higher bio-prevalance of MAP in healthy small ruminants in different animal herds of Odisha, which can’t be ignored. The real difficulty of early detection of MAP is the slow development (2-4 yrs) of disease and present study showed the usefulness of multiple tests for effective screening of animals. The control of Johne’s disease not only improves the animal productivity but also reduces the threat to human population.

Authors are thankful to National Agricultural Science Fund (NASF) for financial assistance and Dean, College of Veterinary Science and Animal Husbandry, OUAT, Bhubaneswar for granting of funds and Head, Animal Health Division, CIRG, Makhdoom for the technical assistance. I would like to render my hearty thanks to Dr. Hemakanta Sahu and his teammates for all the support and assistance provided to me during the time of sample collection from different areas of Odisha.

No conflict of interest to declare.