Expression profiles of different cytokine genes in peripheral blood mononuclear cells of goats infected experimentally with native strain of Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (ParaTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic enteritis of ruminants and may contribute to Crohn's disease in humans. Key features of host immunity to MAP infection include an early pro-inflammatory (Th1-like) response that eventually gives way to a predominant anti-inflammatory (Th2-like) response. Many studies have been conducted to understand the underlying mechanism of misdirected host immune response, however, these studies mainly focused on cattle. The present study is the first attempt to test the hypothesis of shift in Th1 to Th2 like responses during the progression of ParaTB in caprine species (small ruminant). Ten healthy male kids (<6 months old) of the same breed were selected for this study. Of the 10 kids, 6 were experimentally infected with native strain (S5) of MAP ("Indian Bison Type") and the remaining 4 kids were control. Kids were monitored for a period of 12 months post infection (MPI) and were tested for establishment of infection. Expression levels of IFNG, IL2, IL12, IL4, and IL10 genes were estimated before infection and at 4, 8, and 12 MPI in stimulated peripheral blood mononuclear cells (PBMCs) of infected and control kids. The study demonstrated the expression of IFNG and IL2 as classic Th1-like pro-inflammatory signatures; whereas, IL10 exhibited itself as classical Th2-like signature. The study also reports unexpected lowered expression of the IL12 gene simultaneously with increased expression of IFNG, lowered expression of the IL2 gene (compared to IFNG), and suppressed expression of the IL4.

Effect of genetic variation in the MHC Class II DRB region on resistance and susceptibility to Johne's disease in endangered Indian Jamunapari goats.

The pathogenesis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is complex and has not been completely understood yet. In the present study, we analysed the polymorphism in the exon-2 of the caprine major histocompatibility complex (MHC) Class II DRB region and its association with resistance or susceptibility to JD. A total of 203 Jamunapari goats, which is an Indian endangered breed highly susceptible to JD, kept at a single farm were studied. On the basis of clinical signs, microscopic examination, faecal culture, ELISA and diagnostic PCR, 60 and 143 goats were classified as resistant and susceptible to JD, respectively. PCR-based restriction fragment length polymorphism (PCR-RFLP) with two enzymes, PstI and TaqI, was used to assess variation in the DRB gene(s) in all 203 goats studied. Two di-allelic single nucleotide polymorphisms (SNPs), here referred as 'P' and 'T', were tested. In each of them, three genotypes were found in the group analysed. The minimum allele frequencies (MAFs) were 0.233 and 0.486 for the P and T SNPs, respectively. Statistically significant associations between alleles, individual genotypes and composed genotypes of both SNPs were found. The frequency of p and t alleles, of individual pp and tt and of composed pptt genotypes were significantly higher (P(corr) < 0.001) in the 'resistant' group as compared to the 'susceptible' group, while the P and T alleles were associated with susceptibility (P(corr) < 0.001). In heterozygous genotypes, susceptibility was dominant over resistance. The effects of both SNP on resistance and susceptibility were comparable and composed heterozygous genotypes showed intermediate levels of susceptibility in terms of the odds ratio and P-values calculated.

Age induced interactions between heart rate variability and systolic blood pressure variability using approximate entropy and recurrence quantification analysis: a multiscale cross correlation analysis.

The purpose of this study is to study the effect of age on the correlation between heart rate variability (HRV) and blood pressure variability (BPV). To meet this end, multi-scale cross correlation (CC) analysis of HRV and systolic blood pressure variability (SBPV) was performed. The Approximate Entropy (ApEn) and Recurrence Quantification Analysis (RQA) derived indices, calculated from RR interval series (RRi) and systolic blood pressure (SBP) series at multiple temporal scales, are the basis of this CC analysis. For the computation of ApEn and RQA indices, the tolerance threshold (r) is chosen by either: (i) selecting any arbitrary value (0.2) within the recommended range (0.1-0.25) times standard deviation (SD) of time series, and (ii) taking the 'r' (ropt) corresponding to maximum ApEn (ApEnmax) as tolerance threshold. It is found that (i) at each time scale (τ), a lower SD is observed when indices are computed using ropt than [Formula: see text] (r0.2), for RRi as well as SBP series, (ii) descriptive indices of RRi are found significant (p < 0.05) at all scales (τ), however for SBP, these are found insignificant (p > 0.05) at most of the scales, (iii) CC values of descriptive statistics viz., mean and SD are not significant (p > 0.05) irrespective of τ, barring τ = 1, (iv) CC values of ApEn and RQA indices, found using ropt, are found significant (p < 0.05) and provide enhanced stratification at τ = 1, 2 and 3, whereas this significant correlation and strong classification is missing for indices calculated using r0.2, and (v) Lastly as τ increases, ApEn and RQA indices, computed with ropt, reverse their trend but manage to provide significant difference in elder and younger subjects. It is concluded that HRV and SBPV interactions gets altered with age. Descriptive indicators however are not enough to capture these changes. These complex interactions can only be deciphered using complexity-based methods such as approximate entropy and that too at the multiple scale level.

Strain diversity within Mycobacterium avium subspecies paratuberculosis--a review.

Mycobacterium avium subspecies paratuberculosis (MAP), is the etiological agent of Johne's disease (or paratuberculosis) in animals and has also been linked with Crohn's disease of human beings. Extreme fastidious nature of the organism (MAP) has hampered studies on diversity within the organism. Studies based on phenotypic properties like growth rate, pigmentation, lipid profile etc., are unable to provide complete information on diversity of MAP organism in nature. However, with the advent of molecular assays (IS900 RFLP, PFGE, IS1311 PCR-REA, SSR typing, VNTR typing etc.) in last 2 decades, progress has been made to differentiate MAP strains. MAP isolates have been classified into various types and subtypes using these molecular tools. Optimization of these typing assays has led to generation of new information about MAP strains, subtypes, their comparative genomics, relative evolution, comparative virulence etc. Knowledge of strain diversity is important for better understanding of molecular and sero-epidemiology, infection and patho-biology, vaccine development and planning control strategies. The present review provides available information on MAP strains, ho st adaptations, their virulence,comparative genomics, relative genetic evolution and differentiation.

Genotype profiles of Mycobacterium avium subspecies paratuberculosis recovered from suspected and Crohn's disease patients in India.

Present study aimed to genotype Mycobacterium avium subspecies paratuberculosis (MAP) recovered from suspected and Crohn' s disease patients. A total of 32 MAP and DNA (directly from clinical samples) recovered from human origin were genotyped using IS 1311 PCR-REA. Isolates were cultured from stool, biopsies and blood clots of Crohn's disease patients, and stool samples of suspected (animal attendants, lab workers etc). Of the 32 MAP isolates belonging to 28 human beings, majority (84.3%) were genotyped as 'Bison type', while 21.7% were of 'cattle' and none was 'sheep' genotype. Study first time reports distribution of 'Cattle' and 'Bison type' 'genotypes in suspected and Crohn's patients on pilot scale in India. 'Bison type' genotype was predominant in the surveyed human population.

Comparative potential of modified indigenous, indigenous and commercial ELISA kits for diagnosis of Mycobacterium avium subspecies paratuberculosis in goat and sheep.

In the present study, modified indigenous ELISA kit (kit 1) was compared with indigenous ELISA kit (kit 2) and commercial ELISA kit (kit 3) for its sensitivity and specificity with respect to faecal culture for diagnosis of Johne's disease in goats and sheep under natural conditions. Of the 64 positive animals, serum of 42.1, 48.4 and 18.7% animals yielded positive infection in kit 1, 2 and 3, respectively. Specificity of kit 1 (95.1%) was maximum followed by kit 3 (93.7%) and kit 2 (83.4%). Kit 1 showed superior diagnostic potential than the other two kits. Kit 1 may be used as single screening test regimen for diagnosis of MAP infection in the population of goats and sheep in India.

A unified non-linear approach based on recurrence quantification analysis and approximate entropy: application to the classification of heart rate variability of age-stratified subjects.

This paper presents a unified approach based on the recurrence quantification analysis (RQA) and approximate entropy (ApEn) for the classification of heart rate variability (HRV). In this paper, the optimum tolerance threshold (ropt) corresponding to ApEnmax has been used for RQA calculation. The experimental data length (N) of RR interval series (RRi) is optimized by taking ropt as key parameter. ropt is found to be lying within the recommended range of 0.1 to 0.25 times the standard deviation of the RRi, when N ≥ 300. Consequently, RQA is applied to the age stratified RRi and indices such as percentage recurrence (%REC), percentage laminarity (%LAM), and percentage determinism (%DET) are calculated along with ApEnmax, [Formula: see text], [Formula: see text], and an index namely the radius differential (RD). Certain standard HRV statistical indices such as mean RR, standard deviation of RR (or NN) interval (SDNN), and the square root of the mean squared differences of successive RR intervals (RMSSD) (Eur Hear J 17:354-381, 1996) are also found for comparison. It is observed that (i) RD can discriminate between the elderly and young subjects with a value of 0.1151 ± 0.0236 (mean ± SD) and 0.0533 ± 0.0133 (mean ± SD) respectively for the elderly and young subjects and is found to be statistically significant with p < 0.05. (ii) Similar significant discrimination was obtained using [Formula: see text] with a value of 0.1827 ± 0.0382 (mean ± SD) and 0.2248 ± 0.0320 (mean ± SD) (iii) other significant indices were found to be %REC, %DET, %LAM, SDNN, and RMSSD; however, ApEnmax was found to be insignificant with p > 0.05. The above features of RRi time series were tested for classification using support vector machine (SVM) and multilayer perceptron neural network (MLPNN). Higher classification accuracy was achieved using SVM with a maximum value of 99.71%. Graphical abstract.

Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.

OBJECTIVES: To investigate and characterize Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease, attendants of animals with suspected infection, and healthy humans, using multiple diagnostic tests. METHODS: A total of 119 samples (35 stool, 76 serum, three blood clots, and five biopsies) were collected from five patients with Crohn's disease, eight attendants of animals with Johne's disease, and 93 apparently normal control subjects (Agra region) from North India. Samples were screened for the presence of MAP by smear examination, culture of stool, blood clot and biopsies, and ELISA. Colonies obtained by culture were further characterized using polymerase chain reaction (PCR) with IS900 MAP-specific primers. RESULTS: Using all diagnostic modalities, MAP and/or MAP antibodies were identified in 100% (5/5) of subjects with Crohn's disease; 75.0% (6/8) of attendants of MAP infected animals were positive and 38.0% (27/71) of apparently normal controls were also positive. Most sensitive test was ELISA (100%, 5/5), followed by culture (80.0%, 4/5), and acid-fast staining. Ziehl-Neelsen staining was positive in 37.5% (3/8) of subjects with active animal husbandry practices. In 71 serum samples from control subjects, seroprevalence of MAP was 38.0% using indigenous protoplasmic antigens (PPA) and 36.6% using commercial PPA. Of the serum samples from the Crohn's disease patients, 100% (5/5) were positive by ELISA using indigenous PPA and 40.0% (2/5) were positive by ELISA using commercial PPA. IS900 PCR was used to characterize tiny colonies of MAP that grew extremely slowly on Herrold's egg yolk medium, and of 15 (42.8%) cultures, 14 (93.3%) were typed as MAP. CONCLUSIONS: Paper documented the presence of MAP in all patients with Crohn's disease, in some animal attendants who had the history of working with goat herds infected with Johne's disease and in few normal healthy individuals. Presence of Ziehl Neelsen positive MAP. In the stool of attendants working with MAP-infected animals was unique to humans. ELISA based on antigens derived from indigenous MAP 'bison type' genotype of goat origin was most sensitive modality for screening Crohn's disease patients.

Pathogenic 'Bison-type' Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.

Despite low per-animal productivity of ruminants in developing countries, Johne's disease has not been investigated in buffaloes, which are primarily found in these countries. This is due to lack of expertise, diagnostic kits and priority to production diseases like Johne's disease. Presence of pathogenic Mycobacterium avium subspecies paratuberculosis (Map) was investigated by screening of target tissues (mesenteric lymph nodes and large intestine) by culture and IS 900 PCR, in 50 sacrificed buffaloes. Indigenous ELISA kit originally developed for goats and sheep was standardized in buffaloes and used to estimate sero-presence of Map in 167 serum samples representing population of buffaloes in Agra region of North India. In culture, 48.0% buffaloes were positive from 50 tissues each from mesenteric lymph nodes (34.0%) and large intestine (36.0%). IS 900 PCR was standardized using specific primers (150 C and 921) and 229 bp-amplified product was characteristic for Map. Of the 25 mesenteric lymph nodes, 40.0% were positive in IS 900 PCR. Genomic DNA from Map cultures was successfully amplified from all the 24 isolates (100.0%). Map was further genotyped as 'Bison type' using IS 1311 PCR-REA. Culture of tissues showed high presence of Map in target tissues, despite high culling rate in buffalos in view of high demand of buffalo meat. Specific tissue-PCR provided rapid confirmation of Map infection in sacrificed buffaloes. In tissue-PCR, all the cultures were positive as compared to 40.0% detected directly from tissues. ELISA kit using indigenous protoplasmic antigen was highly sensitive as compared to commercial antigen in detecting Map infection therefore, could be used as 'Herd Screening Test' in buffaloes against Johne's disease. This pilot study first time reports a highly pathogenic 'Bison-type' genotype of M. avium subspecies paratuberculosis from the riverine buffaloes (Bubalus bubalis) of Agra region in North India.

Sero-prevalence of bovine Johne's disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis 'Bison type' genotype of goat origin.

Present pilot study is the first attempt in the country to estimate sero-prevalence of Bovine Johne's disease (BJD) by screening cattle and buffaloes representing large population belonging to farmer's and farm herds in the home tracts (Uttar Pradesh (UP) and Punjab) of Hariana cattle and Murrah buffaloes in North India. Indigenous and in-house plate ELISA kit (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and sheep was standardized in bovines and used for screening. For this study, 33 villages of south and west UP were randomly selected and surveyed from 2001 to 2003. There were 7943 farmer's families having 38,251 livestock, including cattle, buffaloes, goats and sheep (per family 4.8% livestock). Numerically, buffaloes and cattle were 54.7% and 22.1%, respectively. Serum samples were collected from 726 animals (4.2% of 16, 981 livestock with 4375 farmer's families) located in 33 randomly surveyed villages. Serum samples (699), submitted to Epidemiology Department of Veterinary College (Punjab Agricultural University, Ludhiana), in the year 2004 by farmer's and organized farm herds (Buffaloes, 372, Cattle, 327), were screened by this ELISA kit. Soluble protoplasmic antigen was prepared from Map (S 5) 'Bison type' strain isolated from a terminally sick goat with Johne's disease. Of the total 1425 bovine (Buffaloes and cattle) serum samples screened using indigenous ELISA kit, sero-prevalence of Johne's disease was 29.0% (28.6% in buffalo and 29.8% in cattle) in Northern India. State-wise sero-prevalence was 31.9% and 23.3% in UP and Punjab, respectively. In UP, of the 601 randomly sampled buffaloes, sero-prevalence was 40.3% (16.6% in young and 40.9% adults) and 25.5% (10.5% in young and 26.3% adults) in south and west UP, respectively. Of the 125 cattle screened, sero-prevalence was 42.6% (nil in young and 44.4% adults) and 30.0% (nil in young and 30.6% adults) in south and west UP, respectively. Of the 699 serum samples screened from Ludhiana, Punjab, sero-prevalence of BJD was 23.0%. Sero-prevalence was 23.3% (12.1% in young and 24.4% in adults) and 26.9% (27.2% in young and 26.8% in adults) in buffaloes and cattle, respectively. High prevalence of BJD in buffaloes in native tract of Murrah breed, and Hariana breed of cattle correlated with poor per-animal productivity and BJD may be the major cause. Indigenous ELISA kit was rapid, economic and sensitive test for large-scale screening of buffaloes and cattle population against incurable BJD.

Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of bovine Johne's disease (BJD) in lactating Indian dairy cattle.

Present study is the first attempt to evaluate an indigenous milk ELISA with milk culture, standardize milk PCR, estimate lacto-prevalence of Map and genotype Map DNA from milk samples in few Indian dairy herds. In all 115 cows were sampled from 669 lactating cows in six dairy herds from three districts of North India. Fifty milk samples (four herds) were screened by three tests (milk culture, m-ELISA and m-PCR). Lacto-prevalence of Map in four dairy herds was 84.0% (50.0% in fat and 62.0% in sediment). Screening of both fat and sediment increased the sensitivity of culture. Colonies appeared between 45 and 120 DPI. In indigenous m-ELISA, protoplasmic antigen derived from native Map 'Bison type' strain of goat origin was used. Screening of 115 lactating cows by m-ELISA ('herd screening test') detected 32.1% positive lactating cows (lacto-prevalence). Sensitivity of ELISA was 28.5% and 42.8% in single point cutoff and S/P ratio, respectively. Lacto-prevalence of JD was high in dairy herds (66.6-100.0% by culture and 20.0-50.0% by m-ELISA). DDD farm, Mathura had very high (95.8%) and moderate prevalence of Map and lacto-antibodies, respectively. All cows were clinically suffering from JD. Specific IS 900 PCR was standardized in decontaminated fat and sediment of milk samples. DNA isolated from decontaminated pellets was amplified and characteristic 229 bp band was confirmatory for Map. Of the 50 milk samples, 6.0% were positive in m-PCR. The test needs further standardization. Map DNA were genotyped as Map 'Bison type' by IS 1311 PCR-REA. Of the three tests, milk culture was most sensitive followed by m-ELISA and m-PCR. Map DNA isolated from milk samples of dairy cattle were first time genotyped as Map, 'Bison type' in India. High prevalence of Map in milk of dairy herds, posed major health hazard for calves and human beings.

Evaluation of four methods of DNA recovery from Mycobacterium avium subspecies paratuberculosis present in intestine tissue of goats and comparative sensitivity of IS900 PCR with respect to culture for diagnosis of Johne's disease.

Low sensitivity of PCR reaction for detection of Mycoobacterium avium subspecies paratuberculosis (MAP) in tissues and fecal samples is mainly attributed to false negative results. Present study was undertaken to compare four methods of DNA isolation from tissues of infected animals and to determine most sensitive protocol for the recovery of DNA, suitable for IS900 PCR based detection of Johne's disease infection. Method I, the traditional van Soolingen2 method of DNA isolation was adopted for the isolation of DNA from tissues. Method II was modification (hexadecyl pyridinium chloride-HPC treatment) of van Soolingen2 method. Method III was traditional tissue DNA isolation method based on tissue lysis buffer. Method IV was modification of method III (HPC treatment). Using four methods of DNA isolation from 25 intestinal tissues of clinically infected goats, DNA was isolated from 15 (60.0%), 18 (72.0%), 13 (52.0%) and 13 (52.0%) tissues using method I, II, III and IV, respectively. All isolated DNA preparations were positive for MAP in IS900 PCR. HPC treatment enhanced the recovery of DNA from tissues of infected animals using method II. Therefore, method II can improve the diagnosis MAP infection using IS900 PCR.

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India.

Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.

Non-chemical method of DNA recovery and characterization of Mycobacterium avium subspecies paratuberculosis using IS 900 PCR.

In the present study, two methods of DNA isolation-routine, traditional and standard DNA isolation protocol for Mycobacteria (Method 1) and a new non-chemicals and non-enzymes (physical) method (Method 2) of DNA recovery have been compared and evaluated in IS900 PCR for the specific detection of pathogen. Using the new Method 2, DNA has been recovered from few (1 - 3 colonies), extremely minute and stunted colonies. DNA, thus, isolated from these colonies (colonies PCR) and cultured for the first time from the cases of Crohn's disease in human beings, dairy cattle, raw milk and pasteurized commercial milk samples has been characterized in the present study. It is the first report from India.

Mycobacterium avium subspecies paratuberculosis diagnosis and strain typing--present status and future developments.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic gastroenteritis of ruminants and has zoonotic importance. We present here a review of MAP with respect to--(i) present diagnostic techniques and important developments; and (ii) MAP strain-typing tools. A summary of the findings to date is presented, and advantages and disadvantages of each of the methods are compared and discussed.

Evaluation of an indigenous ELISA for diagnosis of Johne's disease and its comparison with commercial kits.

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne's disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis 'Bison Type' strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to 'Gold Standard' fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 µgm / well) as compared to purified commercial protoplasmic antigen (4 µgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne's disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to 'Gold standard' fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne's disease. The study reports high prevalence of Johne's disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne's disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.

'Bio-load' and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne's disease: a survey of 28 years (1985-2013) in India.

Bio-load and bio-profile of Mycobacterium avium subspecies paratuberculosis was studied in the domestic livestock population of the country. Of the 23,429 farm and farmer's animals screened, average bio-load was 23.3% (Period of study; 28 years for goats; 13 years for sheep, cattle and buffaloes). Species-wise, bio-load was 20.1, 32.7, 39.3 and 28.3% in goats, sheep, cattle and buffaloes, respectively. Bio-load was significantly lower in time period A (P < 0.001) and B (P < 0.03), compared with period C. Geographical zone-wise, bio-load of MAP was significantly higher (P < 0.05) in Central zone compared with South, West, East and North zones. Bio-load in 11 states ranged from 16.2 to 87.8%. Of 8450, 5643, 8185 and 1151 samples screened by microscopy, culture, indigenous ELISA and IS900 blood PCR, 20.0, 10.6, 35.1 and 26.6% samples were positive, respectively. Bio-load was 32.8 and 31.6% in farm and farmer's goats and sheep, respectively, and 62.1% in farmer's cattle. MAP bio-load was also monitored in four farm units (three goats and one sheep) for breed improvement and three farm goats units for experimental purposes at Central Institute for Research on Goats in Mathura district. Of the 8025 goats and 1525 sheep that died from 1988 to 2013, 10.9 and 3.0% deaths were due to JD, respectively. On the basis of JD and suspected JD, 10.0 and 28.4% goats and 2.2 and 40.9% sheep, respectively were culled from the farm units in 25 years. Microscopic examination of 214 tissues (mesenteric lymph nodes and intestines) of 107 animals, it was observed that bio-load of MAP was high (25.0-60.0%) in farm animals. 'Indian Bison Type' was the dominant biotype, irrespective of domestic livestock species and the geographical zone.

Evaluation of "Indigenous Absorbed ELISA Kit" for the Estimation of Seroprevalence of Mycobacterium avium Subspecies paratuberculosis Antibodies in Human Beings in North India.

In present pilot study aimed to estimate, presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in the human serum samples originating from North India using "Indigenous absorbed ELISA kit" (ELISA kit). The phase I, "ELISA kit" was optimized using protoplasmic antigen from native isolate of MAP "Indian Bison type" recovered from the biopsies of Crohn's disease patients. The phase II, sensitivity and specificity of the kit were estimated as 40.0 and 83.3%, respectively, when evaluated in 40 human serum samples (5 Crohn's disease and 22 ulcerative colitis patients and 13 healthy human subjects) with defined MAP status with respect to stool culture. Seroprevalence of MAP antibodies was higher in CD patients (80.0%) as compared to ulcerative colitis patients (4.5%) and normal human subjects (15.3%). The phase III, seroprevalence of MAP antibodies was estimated as 23.4%, on the basis of the screening of 452 human serum samples (without history) from different geographical regions of North India. Region-wise, 34.0, 33.3, 32.8, 25.0, 23.0, 17.7, and 12.5% samples were positive from the states of Punjab, Uttarakhand, New Delhi, Himachal Pradesh, Haryana, Uttar Pradesh and Jammu and Kashmir, respectively. Study reported moderately higher presence of MAP antibodies in human population, which necessitates programs to reduce the bioburden of MAP in the environment and in animal population.

High prevalence of Mycobacterium avium subspecies paratuberculosis ('Indian bison type') in animal attendants suffering from gastrointestinal complaints who work with goat herds endemic for Johne's disease in India.

OBJECTIVES: In this study we aimed to estimate the prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) in animal attendants who were chronic colitis patients or who had inflammatory bowel disease and were suspected for Crohn's disease; these animal attendants worked with goat herds endemic for Johne's disease. Microscopic examination and culture tests were used. For comparison purposes a group of healthy human subjects (not suffering with colitis) was also screened. METHODS: Stool samples obtained from 98 human subjects (58 animal attendants suspected for Crohn's disease and 40 healthy humans) were screened for the presence of MAP by microscopic examination and culture. Of the 58 animal attendants screened, 38 had abdominal pain, 29 had suffered episodes of diarrhea, 39 had experienced weight loss, 27 had fever, and 32 had a history of raw milk consumption. Animal attendants had had contact of variable duration with goat herds endemic for Johne's disease (1-5, 6-10, 11-15, and >15 years). Forty stool samples from healthy humans with no symptoms/history of contact with animals were also screened. IS900 PCR and IS1311 PCR restriction endonuclease analysis were used to characterize and genotype the MAP colonies. RESULTS: MAP was recovered from 34 of the 98 human subject stool samples (34.7%). Of the 98 samples, 16.3% (n=16) were acid-fast. None of the 40 healthy human subjects were positive for MAP by microscopy, but five (12.5%) were positive for MAP by culture. Of the 58 animal attendants, 16 (27.6%) were positive by microscopy and 29 (50%) were positive by culture. MAP were recovered from 68.4% of animal attendants with abdominal pain, 72.4% of those with diarrhea, 71.8% of those with weight loss, 44.4% of those with fever, and 46.9% of those who had a history of raw milk consumption. Of the 29 culture-positive animal attendants, 48.3% had worked for >15 years, 27.6% for 11-15 years, 20.7% for 6-10 years, and 3.4% for 1-5 years with goat herds endemic for Johne's disease. Of the 34 culture isolates, 28 (82.4%) showed good quality DNA on agarose gel and were positive by IS900 PCR. Of the 28 IS900-positive DNA samples, 23 (82.1%) were genotyped as 'Indian bison type' and five (17.9%) as 'cattle type'. CONCLUSIONS: The prevalence of MAP was higher in attendants suffering from gastrointestinal problems who worked with goat herds endemic for Johne's disease, than in humans with no history of contact with animals. The risk of developing gastrointestinal problems with clinical symptoms indistinguishable from inflammatory bowel disease was higher in humans who were in contact with goat herds endemic for Johne's disease as compared to healthy humans, and the risk was correlated with the duration of association with the endemic goat herds.

On the evolution of 'Indian Bison type' strains of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in animals, has also been linked with Crohn's disease in human beings. Lack of indigenous diagnostics and vaccine hampered control of JD in India. Designing effective control strategies require thorough understanding of the etiological agent at phenotypic and molecular levels. On the basis of cultural phenotypes and IS1311 PCR-REA typing, MAP strains have been genotyped as 'Cattle type', 'Sheep type' and 'Bison type'. Information exists on genetic differences and comparative evolution of 'Cattle type' and 'Sheep type' strains after divergence from M. avium; however, emphasis has been little on 'Bison type' strains. Recently, a new 'Indian Bison type' genotype has been reported as principal strain infecting different animal species and human beings in India. The study analyzed few genetic markers to have inferences on the molecular evolution of native MAP isolates belonging to 'Bison type' genotype. Results pointed towards recent evolution of 'Bison type' genotype.