Screening of Human Population for Type 1 Diabetes and Mycobacterium avium Subspecies paratuberculosis from Chattarpur District of Madhya Pradesh

| Mycobacterium avium subspecies paratuberculosis, the cause of granulomatous enteritis in ruminants, has also been associated with Inflammatory Bowel disease or Crohn’s disease in human population. Present study aimed to investigate bio-presence of MAP in suspected and confirmed cases of Type 1 Diabetes in clinical samples (blood and serum) of human beings from Chattarpur district of Madhya Pradesh. Screening of 88 serum samples for the presence of MAP using ‘Indigenous ELISA’, 34 (38.6%) were positive for MAP infection. Whereas of the 71 blood samples screened, 28 (39.4%) were positive in IS900 PCR. Screening of 19 serum and 16 blood samples from 20 confirmed patients of ‘Type 1 diabetes’, 31.5 and 43.7% were positive by ‘Indigenous ELISA’ and IS900 PCR, respectively. Comparison of Indigenous ELISA and IS900 PCR revealed fair agreement between the two tests. Bio-typing of DNA of 8 positive blood samples by IS1311 PCR-RE revealed presence of ‘Indian Bison type’ biotype. Presence of MAP bacterimia in the patients suffering from Type 1 Diabetes added to the existing knowledge that MAP may provide foundation for establishing diabetes in human subjects. This is the first report of presence of MAP in human Type 1 Diabetes patients from India. ‘Indian Bison Type’ biotype in domestic livestock suggests a link and interspecies transmission through food chain.


Advances in
milk powder etc) world-wide (Grant et al., 2001;Ayele et al., 2005;Shankar et al., 2010).In India, raw milk serves as base of many Ayurvedic medicines.Recently role of MAP has been reported in different kinds of human diseases such as Type-1 Diabetes (Sechi et al., 2008), autoimmune thyroiditis (D'Amore et al., 2010), multiple sclerosis (Cossu et al., 2013), autism (Dow, 2011), sarcoidosis (EL-Zaatari et al., 1996) and autoimmune arthritis (Moudgil et al., 1997).Recent years witnessed a surge in cases of Type-1 Diabetes particularly the 'Type 1 diabetes mellitus', a chronic disease in which insulin producing beta cells of the pancreas are selectively destroyed by T lymphocyte infiltration, in human beings.Disease is the second most common chronic disease during childhood.Annual incidence of the Type 1 Diabetes (per 100,000) is consistently increasing and the highest being in European countries (32.5), followed by Non-Europeans (14.4), Maori (13.9),Pacific Islanders (15.4) and others (13.5) (Derraik et al., 2012).In Northern India, occurrence of Type 1 Diabetes was found to be 10.20 per 100,000 population with a higher prevalence in urban (26.6/100,000) as compared to rural areas (4.27/100,000).In the age group of 5-16 years, prevalence is 22.22/100,000, while in 0-5 years age group prevalence is 3.82/100,000 (Kalra et al., 2010).Susceptibility to Type 1 Diabetes is inherited, but mode of inheritance is not clearly understood.Several loci, primarily HLA and environmental factors might be responsible for manifestation of disease (Concannon et al., 2009;Van Belle et al., 2011).MAP has also been indicated as an environmental trigger that might contribute to Type-1 Diabetes pathogenesis (Sechi et al., 2008;Naser et al., 2013). Singh et al. (2014) reported high burden of MAP in different kinds of infectious and non-infectious disease conditions e.g., diabetes, liver disorders, anemia, thyroid disorder, tuberculosis, typhoid, abdominal disorders, inflammatory illness and ion imbalance in human population from North India.The study aimed to screen human population for Type 1 Diabetes and Mycobacterium avium subspecies paratuberculosis in Chatarpur district of Madhya Pradesh state in India.

ColleCtion of SampleS
Informed consent from patients as well as other necessary clearances were procured before blood samples were drawn.A total of 111 human samples (88 serum and 71 blood) were collected from a 'health camp' organized by Gwalior Medical College at Chattarpur district of Madhya Pradesh to screen human population for diabetes and MAP infection.Of the 111 suspected human beings screened for diabetes, only 20 (18.0%) were positive using commercial kits (AC-CU-CHEK blood glucose monitoring system, Roche).

inDigenouS eliSa Kit
Human serum samples were screened by 'Indigenous ELISA kit ' (Singh et al., 2007) for presence of MAP infection.Absorbance was read at 450 nm in iMark ELISA reader (BioRad).Defined positive and negative were simultaneously run.Optical density (OD) values were converted to sample to positive (S/P) ratios as per Collins (2002) to determine the status of MAP infection.Serum samples in positive and strong positive categories were considered positive for MAP infection.Ratio between mean OD of positive and negative control (≥ 4 times) was critical for calculating S/P ratio and categories were (0.00-0.09, negative; 0.10-0.24,suspected; 0.25-0.39,low positive; 0.40-0.99,positive; and 0.61-0.80,strong positive) as per Collins (2002).iS900 pCr MAP DNA was extracted from blood samples (n=14) and was subjected to screening by specific IS900 PCR following the protocol of Singh et al. (2010b).Briefly, in a volume of 12.5μl of 2X red dye PCR master mix (Genei), 1μl each of forward (10 pmol/μl) and reverse primer (10 pmol/μl), 7.5μl of nuclease free water and 3μl template DNA were added (25μl total volume).Thermal cycling conditions were: initial denaturation (94°C for 5 min), followed by 35 cycles of denaturation (94°C for 30 sec), annealing (64°C for 30 sec), extension (72°C for 30 sec), and a final extension (72°C for 10 min).Product size of 413 bp was considered positive after separation on 1.5% agarose gel electrophoresis.MAP IS900 primer sequences used as per Millar et al. (1996) were: P90 5'-GAAGGGTGTTCGGGGCCGTCGCTTAGG -3' (Forward primer) and P91 5'-GGCGTTGAG-GTCGATCGCCCACGTGAC -3' (Reverse primer).(2005).Briefly, in a volume of 15μl of 2X red dye PCR master mix (Genei), 1μl each of forward (10 pmol/μl) and reverse primer (10 pmol/ μl), 10μl of nuclease free water and 3μl of template DNA were added (total volume 30μl).Thermal cycling conditions were: initial denaturation (94°C for 5 min), followed by 35 cycles of denaturation (94°C for 30 sec), annealing (62°C for 30 sec), extension (72°C for 1 min), and a final extension (72°C for 10 min).Amplicon size of 608 bp on 1.5% agarose gel with ethidium bromide further confirmed presence of MAP infection.

StatiStiCal analySiS
Proportional agreement (Kappa coefficient) between the tests was compared as per Arizmendi and Grimes (1995).

inDigenouS eliSa Kit
Of 88 serum samples screened, 34 (38.6%) were positive for the presence of MAP infection.Of the 20 confirmed 'Type 1 Diabetes' patients, 19 serum were collected and screened and of which 6 (31.5%) were positive for MAP infection by 'Indigenous ELISA' (Table 1 and 2).Comparative evaluation of 'Indigenous ELISA' with IS900 PCR showed that 14.7% human subjects were positive by 'Indigenous ELISA kit' as compared to IS900 PCR (20.5%).However, cumulatively, 16 (23.5%)human subjects were positive by the two tests.Proportional agreement between 'Indigenous ELISA' and IS900 PCR was substantial (64.7%) (Table 3 and 4).
Of 28 IS900 PCR positive samples, eight were subjected to IS1311 PCR and were positive (Figure 2).Bio-typing of 8 positive samples by IS1311 PCR-REA showed presence of highly pathogenic 'Indian Bison type' biotype (Figure 3).ings (Chiodini et al., 1984;Singh et al., 2010aSingh et al., , 2011Singh et al., , 2014)).Recent Indian studies report sharp increase in bioload of MAP in domestic livestock species (Singh et al., 2013;Singh et al., 2014).A sub-clinically infected animal excrete huge quantities of bacilli in feces, milk, semen etc., and MAP is known to sur  2012) reported contamination of natural resources (soil and water) with MAP in North India.It is therefore important to restrict contamination of environment and natural resources by animal and human excreta laden with MAP in order to reduce transmission to animal and human population.To reduce contamination of human population with MAP, it is essential to reduce infection of MAP in animals, especially domestic livestock.Association between MAP and chronic inflammatory bowel disease (Crohn disease) has been reported and found to persist in cell wall-deficient form, escaping clearance by the host immune system (Sechi et al., 2008).Other reports suggested association of MAP with Type I diabetes (autoimmune disease) in patients from Sardinia (Italy), which is a MAP endemic and genetically isolated region (Rani et al., 2010;Cossu et al., 2011;Cossu et al., 2013, Bitti et al., 2012, Masala et al., 2014) et al., 2008), which also has high rate of MAP infection in their livestock (Rani et al., 2010) may not be true in India since large variability exists both in Indian population with respect to animals (species and breeds) and human beings.
Economic significance of MAP in dairy enterprise in general and domestic livestock in particular, combined with contamination of milk and milk products (Public Health significance), risk of human infection and increasing role of MAP in the development of Crohn's disease, type 1 diabetes and other incurable diseases in human beings highlight the need to minimize chances of entry of bug in to food chain.Present findings are first report from India supporting the hypothesis of probable association of MAP and type 1 diabetes suggesting that there is sufficient ground to warrant focus on MAP and its possible involvement in triggering Type 1 Diabetes.
DNA were subjected to IS1311 PCR using forward (M56) and reverse (M119) primers as per Sevilla et al.
Restriction endonuclease analysis (PCR-REA) was carried out employing endonuclease enzymes (HinfI and MseI) as per Sevilla et al. (2005).Briefly, REA reaction was performed in a 30μl volume containing 20μl positive IS1311 PCR product, 5μl reaction 10X buffer and 1μl (5U) each of HinfI and MseI endonucleases (Fermentas), and reaction mixture incubated at 37°C for 2hrs.Digested products were visualized on 2% agarose gel electrophoresis and biotypes (band patterns) were interpreted as described by Whittington et al. (2001).

*
Positive and strong positive in S/P ratio taken as positive (38.6%);Figures in parentheses are percentage.

Table 1 :
Comparative evaluation of S/P ratios of the results of Indigenous ELISA

Table 2 :
Screening of type 1 diabetes and healthy suspects for MAP infection by Indigenous ELISA kit and IS900 PCR

Table 3 :
Comparative evaluation of Indigenous ELISA kit and IS900 PCR for the presence of MAP infection *Figures in parentheses are percentage; Agreement between 'Indigenous ELISA kit' and IS900 PCR -64.7%

Table 4 :
Comparative evaluation of Indigenous ELISA kit and IS900 PCR for the presence of MAP infection in confirmed type 1 diabetes humans *Figures in parentheses are percentage; Agreement between 'Indigenous ELISA kit' and IS900 PCR -50.0%

in Animal and Veterinary Sciences November 2014 | Volume 2 | Issue 11 | Page 617
could be a population specific phenomenon, highly dependent on genetic repertoire and the environment of susceptible population.Singh et  al. (2014)also reported difference in sero-prevalence MAP in human population between two adjacent districts; Agra (14.2%) and Mathura (35.4%), which may be due to chance, since population in two regions is very similar except Agra is growing metropolitan city and Mathura is still rural type.Contention of Rani et al. (2014) that high genetic susceptibility with respect to presence of SLC11A1 gene in type 1 diabetes affected population of Sardinia (Pugazhendhi