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.

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.

'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.

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.

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.

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.

Genotype diversity in Indian isolates of Mycobacterium avium subspecies paratuberculosis recovered from domestic and wild ruminants from different agro-climatic regions.

Fecal samples of 708 domestic and 27 wild ruminants from seven districts of three states submitted to Microbiology Laboratory from 2004 to 2008 were screened for the presence and genotype diversity of Mycobacterium avium subspecies paratuberculosis (MAP) stusing modified microscopic examination and culture tests. MAP colonies were characterized and genotyped by IS900 PCR and IS1311 PCR-REA, respectively. In microscopic examination and culture, 18.7 and 31.2% sampled were positive, respectively. Percent positivity varied in different species and agro-climatic regions. Of the 151 DNA recovered from 230 MAP isolates, 94.7% (143) and 5.2% (8) were genotyped as 'Indian Bison type' and 'Cattle type', respectively. Cattle and buffaloes were infected with both the genotypes, whereas goat and sheep were infected exclusively by 'Indian Bison type'. Study showed low diversity in MAP genotypes and 'Indian Bison type' was the predominant genotype shared by different species, breeds and agro-climatic regions.

Is Mycobacterium avium subsp. paratuberculosis, the cause of Johne's disease in animals, a good candidate for Crohn's disease in man?

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease or paratuberculosis, a gastro intestinal inflammatory condition in ruminants and other animals, which is similar to Crohn's disease (CD) that occurs in man. The role of MAP in the causation of CD has been under intense investigation in the last few decades. This review summarizes the status of MAP in animals and the food chain and its association with CD in man.

Therapeutic Effects of a New "Indigenous Vaccine" Developed Using Novel Native "Indian Bison Type" Genotype of Mycobacterium avium Subspecies paratuberculosis for the Control of Clinical Johne's Disease in Naturally Infected Goatherds in India.

Therapeutic efficacy of an "Indigenous vaccine" has been evaluated with respect to a commercial vaccine (Gudair, Spain), for the control of clinical Johne's disease (JD) in naturally infected goatherds. Seventy-one goats (JD positive) were randomly divided into 3 groups ("Bison", "Gudair" and "Sham-immunized"). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of M. paratuberculosis (MAP) in feces, internal condition and lesions, as well as humoral and cell-mediated immune responses for 210 days. Study showed marked overall improvement in physical condition of vaccinated goats and average body weight gain was significantly higher (P < .05) in "Bison" group as compared to "Sham-immunized" goats. Mortality due to JD was significantly (P < .05) lower in vaccinated groups than in "sham-immunized". Morbidity rates (due to diarrhea and weakness) were lower in "Bison" group as compared to other groups. Died goats from vaccinated groups showed regression of gross JD lesions and regeneration of fat layer around visceral organs while "Sham-immunized" goats exhibited frank lesions. Vaccinated goats had higher protective CMI response and also higher antibody titer for the trial period as compared to "Sham immunized". Both vaccines also reduced shedding of MAP in feces significantly (P < .05). Though the two vaccines effectively restricted the severity of clinical symptoms of JD, however "Indigenous vaccine" was superior in many respects.

Diagnostic Application of IS900 PCR Using Blood as a Source Sample for the Detection of Mycobacterium avium Subspecies Paratuberculosis in Early and Subclinical Cases of Caprine Paratuberculosis.

Efficacy of IS900 blood PCR was evaluated for the presence of MAP infection. Serum, fecal, and blood samples of kids, young, and adult goats from farm and farmer's herds in Mathura district were also screened by ELISA, microscopy and culture. Of 111 goats (kids: 40, young: 14, adults: 57) screened, 77.5% were positive by blood PCR. Of 76 goats, 90.8% (kids: 87.5% and adults: 94.4%) were positive by PCR. From 21 kids and 14 young goats, 42.8 and 57.1% were positive. gDNA from goats was genotyped as MAP "Indian Bison type". Of 21 fecal samples of kids examined by microscopy, 66.7% were positive. In ELISA, 9.5 and 57.1% kids were positives as "type I" and "type II" reactors, respectively. Screening 14 young goats by culture of blood clots, 28.6% were positive. Agreement was substantial between PCR and microscopy. It was fair and moderate when PCR and microscopy were compared with type I and type II reactors, respectively. Presence of MAP in non-clinical kids and young goats indicate early or subclinical infection. Blood PCR was rapid, sensitive, and specific assay for detection of MAP in any stage (early, subclinical, and clinical) and age (kids, young, and adult) of goats.

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.

Presence, characterization, and genotype profiles of Mycobacterium avium subspecies paratuberculosis from unpasteurized individual and pooled milk, commercial pasteurized milk, and milk products in India by culture, PCR, and PCR-REA methods.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, a chronic enteritis evocative of human inflammatory bowel disease. In industrialized countries MAP has been cultured from pasteurized milk, compounding the increasing concern that MAP may be zoonotic. The purpose of this study was to evaluate commercially available unpasteurized and pasteurized milk and its products for the presence of viable MAP or MAP DNA from an area of northern India with a population of 150 million people. METHODS: We studied 43 samples (16 unpasteurized, 27 pasteurized) purchased in Mathura, Agra, or New Delhi, for the presence of MAP by culture or by PCR for IS900 MAP DNA. Positives results were confirmed as MAP by restriction endonuclease analysis and/or DNA sequencing. RESULTS: Colonies appeared in 1.5-20 months post-inoculation. Of the unpasteurized samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were positive for IS900 MAP DNA. Of the pasteurized samples, 67% (18/27) were MAP culture-positive and 33% (9/27) were IS900-positive. Subsequently, 100% (25/25) of the cultured colonies were IS900 and IS1311 MAP DNA-positive. CONCLUSIONS: This is the first report from a developing country of MAP cultured from both pasteurized and unpasteurized milk and milk products. Thus we corroborate the presence of viable MAP in the food chain reported from industrialized countries. With the increasing concern that MAP may be zoonotic, these findings have major implications for healthcare in India. The decreased sensitivity in detecting MAP DNA by PCR directly from milk should be ascribed to our employing only one set of PCR primers.

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.

Wildlife (Boselaphus tragocamelus)-small ruminant (goat and sheep) interface in the transmission of 'Bison type' genotype of Mycobacterium avium subspecies paratuberculosis in India.

Information on Mycobacterium avium subspecies paratuberculosis (MAP) genotypes infecting different animal species in India is limited. Presence of MAP was investigated in free ranging antelopes (locally known as Nilgai/blue bulls/Boselaphus tragocamelus) using direct microscopy, culture, IS900 PCR and IS1311 PCR-REA. IS900 elements of MAP from Nilgai and previously isolated from goats were sequenced and compared to establish inter-species transmission between free ranging Nilgai and closed farm herds and flocks of goats and sheep sharing common grazing and water resources. Fecal samples were collected from two geographical regions (Mathura and Kanpur Dehat districts) separated by 300km, in North India. Of the 42 fecal samples cultured, MAP colonies were recovered from 23.8% samples (Nilgai). Of the 10 positive fecal samples, two were in 'Super shedder' (>1000cfu/g) category and rest were moderate (<10-100cfu/g) shedders. None of the Nilgai from Kanpur Dehat was positive in culture. The 229bp fragment targeting specific IS900 sequence was amplified from template DNA isolated from all the positive MAP cultures of Nilgai. Using IS1311 PCR-REA, MAP colonies were genotyped as 'Bison type'. Goatherds and a sheep flock located at Central Institute for Research on Goats (CIRG), shared 303.52ha of land (Mathura district of Uttar Pradesh) with Nilgai and were endemic for MAP infection. MAP strains isolated from goats and sheep have been genotyped as 'Bison type'. Nucleotide sequence of the insertion elements (900) from MAP 'Bison type' strain (S5) of goat origin and MAP (B42) from Nilgai showed difference of 2 (1%) base pairs at the 11th and 12th position (Genbank accession number EU130943). Study is first report on sharing (inter-species transmission) of a new 'Bison type' genotype of MAP between free ranging wildlife (Nilgai population) and domestic animals (farm goatherds and sheep flocks) in India.

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.

Genotype profiles of Mycobacterium avium subspecies paratuberculosis isolates recovered from animals, commercial milk, and human beings in North India.

OBJECTIVE: To understand the genetic diversity of Mycobacterium avium subspecies paratuberculosis (MAP) isolates recovered from domestic and wild ruminants, commercial milk, and human beings in North India. METHODS: Genotyping of MAP isolates (N=117) recovered from animals, commercial milk, and human beings in different regions of North India between 1998 and 2007 was carried out using IS1311 PCR-restriction enzyme analysis (REA) and short sequence repeat (SSR) typing (G and GGT repeat loci). RESULTS: Of the 117 MAP isolates recovered from North India, bison-type was the predominant (83.8%) genotype followed by cattle-type (16.2%). Bison-type was the exclusive genotype recovered from goats, sheep, buffaloes, and blue bulls. However, both bison-type and cattle-type genotypes were recovered from cattle, humans, and commercial bovine milk samples. The relative distribution of the two genotypes was different in the different regions. Bison-type was the major genotype at the Central Institute for Research on Goats (CIRG), Akos, Ajmer, and Mathura, whereas, cattle-type was the major genotype from New Delhi and Agra. SSR typing of these isolates revealed that all MAP bison-type isolates had an identical profile (7g4ggt) with respect to G and GGT repeat SSR loci. In this study the sheep-type genotype was not found in North India. CONCLUSIONS: This study is the first from India to report the presence of two kinds of MAP genotypes (cattle-type and bison-type). However, non-reporting of the sheep-type genotype may not mean that it is absent in North India; the use of multiple culture media to recover MAP from clinical samples for future investigations is advised.

Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: genomic insights.

Effective control of paratuberculosis and investigations of potential link to Crohn's disease have been hampered by the lack of effective assays for easy and accurate diagnosis of Mycobacterium avium subspecies paratuberculosis (Map). Map is extremely fastidious and depends on iron chelator (Mycobactin). Map strains from humans and sheep are very difficult to isolate and may require years to emerge. Therefore, small numbers of Map isolates have been maintained in available collections. This situation has limited the study of biodiversity of Map. Though, much is known about environmental and host factors that contribute to paratuberculosis disease, but little is known about bacterial genetic mechanism of infection. Diagnostic and strain typing markers still demand improvements. Complete genome sequence of Map K10 strain is available in public domain for comparative genomics with other mycobacteria and clinical isolates of Map. It is anticipated that the genome sequence will help in carrying molecular diagnosis and strain typing with respect to Map forward at rapid pace. This paper reviews the current diagnostic and strain typing markers, which may be useful in typing of clinical isolates in near future.

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.