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1.
Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis is endemic in the domestic livestock population, still it is not priority for control in the country. First time we used 'multiple assays’ for screening raw milk of 465 goats (farm/farmer's herds) to estimate bio-load and bio-type profile of bacilli. Each sample was screened by six tests and compared their sensitivity and specificity. Of 465 raw milk samples screened, bio-load of bacilli was 65.3% by six assays. Assay-wise bio-load was 49.4 and 62.7% in antigen and antibody detection tests, respectively. Bio-load was 48.8, 46.6, and 13.9% in Indirect Fluorescent Antibody Test (i_FAT), microscopy and IS900 PCR and 39.1, 57.4 and 55.6% in Indirect Enzyme Linked Immuno Sorbant Assay (i_ELISA), Dot Enzyme Linked Immuno Sorbant Assay (d_ELISA) and Latex Agglutination Test (LAT), respectively. Dot-ELISA was most sensitive followed by LAT, i_FAT, microscopy and i_ELISA. Milk DNA samples positive in IS900 PCR on bio-typing using IS1311 PCR_ Restriction Enzyme Analysis (IS1311 PCR_REA) revealed, 72.3% (47/65) were 'Indian Bison Type'. Milk was easy to collect sample and first time we used 'whole milk' as 'test sample' without centrifugation. High bio-load of MAP in milk underlined need for urgent control of disease in lactating goatherds. Bacilli was important 'Milk born' infection and on the basis of sensitivity, specificity, resources and requirements, of the ‘six assays’ most appropriate assay/s (single or in combination) can be chosen for the screening and diagnosis of Johne’s disease in lactating goatherds using whole milk as sample.  相似文献   

2.
Murrah buffaloes, best breed for milk production are native of Haryana state. They contributes significantly to the farmer’s income, livelihood and food (milk and meat) security, in the semi-tropical regions of North India. Johne’s disease though endemic in the domestic livestock of the country, but reports are not available in the buffaloes suffering from morbidity due to progressive weakness and diarrhoea. We estimated the status of JD in diarrhoeic buffaloes and cattle reporting at Veterinary Clinical Complex of Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India, using conventional, serological and PCR assays.141 buffaloes suffering from chronic diarrhoea were screened to estimate sero-prevalence of MAP and 50.0 % young and 53.52 % adult animals were positive. Of 14 cattle screened, none of the young and 66.6 % adult cows were positive. In buffaloes, 66.1 and 6.77 %, fecal samples were positive in microscopy and IS900 PCR, respectively. Sero-prevalence of JD was very high in diarrhoeic buffaloes and cattle from Haryana state of India.Buffaloes positive for Mycobacterium avium subspecies paratuberculosis (MAP) infection had reduced total leukocyte count and lymphocytes.  相似文献   

3.
Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based ‘Nano-immuno test’ capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the ‘nano-immuno test’ in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of ‘nano-immuno test’ with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel ‘nano-immuno test’ makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.  相似文献   

4.
Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSPA 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.  相似文献   

5.
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.  相似文献   

6.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.  相似文献   

7.
Slurry from dairy farms is commonly used to fertilize crops and pastures. This mixture of manure, urine and water can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP) is a major concern. Persistence of MAP in soil and infection of soil Acanthamoeba was evaluated by culture, real-time IS900 PCR, and by staining of amoeba with acid-fast and vital stains comparing soils irrigated with MAP-spiked or control dairy farm slurry. MAP DNA was detected in soil for the 8 month study duration. MAP was detected by PCR from more soil samples for plots receiving MAP-spiked slurry (n = 61/66) than from soils receiving control slurry (n = 10/66 samples). Vital stains verified that intracellular MAP in amoeba was viable. More MAP was found in amoeba at the end of the study than immediately after slurry application. There was no relationship between MAP presence in soil and in amoeba over time. Infection of amoeba by MAP provides a protected niche for the persistence and even possibly the replication of MAP in soils. As others have suggested, MAP-infected amoeba may act like a “Trojan horse” providing a means for persistence in soils and potentially a source of infection for grazing animals.  相似文献   

8.
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9.
Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne’s disease were screened by Ziehl–Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region.  相似文献   

10.
This review underlines the public health significance of ‘Indian Bison Type’ of Mycobacterium avium subspecies paratuberculosis (MAP) and also its potential as ‘zoonotic infection’. In the absence of control programs, bio-load of MAP is increasing and if we take total population of animals (500 million plus) and human beings (1.23 billion plus) into account, the number of infected animals and human beings will run into millions in India. Our research on screening of over 26,000 domestic livestock for MAP infection using 4 different diagnostic tests (microscopy, culture, ELISA and PCR), during last 31 years has shown that the average bio-load of MAP in the livestock population of India is very high (cattle 43%, buffaloes 36%, goats 23% and sheep 41%). ‘Mass screening’ of 28,291 human samples between 2008–2016 revealed also high bio-load of MAP. It has been proved that MAP is not in-activated during pasteurization and therefore live bacilli are continuously reaching human population by consumption of even pasteurized milk and other milk products. Live bacilli have also been recovered from meat products and the environment thus illustrating the potential of MAP as pathogen of public health concern. However, at present, there is inadequate scientific evidence to confirm a conclusive link between MAP infection and Johne's disease in ruminants and some cases of Crohn's disease in human beings.  相似文献   

11.
Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n = 86), buffalo (n = 34), goat (n = 25) and sheep (n = 26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed ‘Bison’ type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. ‘Cattle’ type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of ‘Cattle’ type Map from buffalo and goat species in India.  相似文献   

12.
Swinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in India.  相似文献   

13.
The role of terrestrial vertebrates in the epidemiology of chikungunya disease is poorly understood. We evaluated their exposure and amplification role during the 2006 chikungunya outbreak in the Indian Ocean. Blood samples were collected from 18 mammalian and reptile species from Reunion Island, Mauritius and Mayotte. Among the 1051 samples serologically tested for chikungunya virus (CHIKV), two crab-eating macaques (Macaca fascicularis) and two ship rats (Rattus rattus) proved to be exposed to CHIKV. CHIKV RNA was not detected in 791 analyzed sera. Our results confirm the preferential infection of simian primates and suggest that other vertebrates played a poor or no role in CHIKV transmission during the 2006 outbreak.  相似文献   

14.
Two African buffalo (Syncerus caffer), an eland (Taurotragus oryx) and a waterbuck (Kobus defassa) were intravenously inoculated with Cowdria ruminantium (Kiswani). Amblyomma gemma nymphs were fed on the animals at 3 weekly intervals. Jugular blood was also collected at 3 weekly intervals and inoculated into sheep. Nymphal ticks that fed on one buffalo on days 16 and 37 and on the other buffalo on day 58 after infection transmitted the disease as adults to sheep. Nymphs that were applied to the eland 16 days after infection also transmitted the disease to sheep. No nymphs that had fed on the waterbuck transmitted the disease. This is the first report of transmission of heartwater by Amblyomma gemma from infected wild ruminant species to a susceptible domestic ruminant species.  相似文献   

15.
Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit (Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research.  相似文献   

16.

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.  相似文献   

17.
Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran.  相似文献   

18.
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.  相似文献   

19.
One of the most relevant aspects in the diagnosis of paratuberculosis (Johne’s disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated.For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively.The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.  相似文献   

20.
Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD or paratuberculosis) in animals and has also been implicated in Crohn's disease of humans. It has been shown that MAP is endemic in animal population of India. Understanding of heterogeneity among MAP strains is important both for diagnosis and design control measures. Genotyping and epidemiological investigations revealed that MAP 'Bison type' was the predominant strain infecting domestic ruminant population in India. MAP 'Bison type' has also been reported from USA. A number of comparative genomics studies have been conducted to understand 'Cattle type' and 'Sheep type' strains. However, present study was the first attempt to characterize MAP 'Bison type' S5 using different markers including IS900, ISMAP02, IS1311, LSPs and SSRs. Study showed that MAP S5 is similar to MAP K10 in terms of number of IS900, IS1311 and ISMAP02 elements. There was high sequence similarity for IS900 and ISMAP02 between MAP K10 and MAP S5. However, this study also reported genetic differences between two strains. In some IS1311 loci, TG gap at 64th and 65th position was observed in MAP S5. Further sequencing of few more MAP isolates confirmed that this gap was specific to indigenous MAP 'Bison type' and can be further used as molecular signature. ISMAP02 locus 1 was observed at polymorphic position in MAP S5 compared to MAP K10. MAP 'Bison type' S5 also showed polymorphic profile for LSP(P)4. Polymorphism was also observed in SSRs. This pilot study may form the basis for future epidemiological investigations.  相似文献   

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