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PubMed2004

Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies Paratuberculosis infection in cattle using quarter milk and bulk tank milk samples.

A study was conducted to evaluate sensitivity, specificity, and predictive value of IS900-PCR assay for detection of Mycobacterium avium subspecies paratuberculosis in pooled quarter milk and bulk tank milk. Feces, blood and pooled quarter milk from 1493 lactating cattle on 29 herds were analyzed. Bulk tank milk (n = 29 bulk tanks) samples were also examined. Culture analysis revealed that 10.9%, 2.8%, and 20.6% of fecal, pooled quarter milk samples and bulk tanks were positive for M. avium subsp. paratuberculosis, respectively. While 13.5% and 27.5% of pooled quarter milk samples and bulk tanks were positive by IS900 PCR assay, respectively. Moderate to high antibody titers for M. avium subsp. paratuberculosis were detected in 223 of 1493 (14.4%) cows. Cows positive on fecal culture were taken as true positives relative to which the IS900 PCR assay was evaluated. The sensitivity and predictive value of KELA, pooled quarter milk culture, and IS900 PCR assay increased with lactation age. While the specificity of the tests decreased with increase in lactation age. Overall, the IS900 PCR assay using pooled quarter milk samples had a sensitivity, specificity and predictive value of 0.87, 0.95 and 0.71, respectively. The IS900 PCR assay using bulk tank milk had poor sensitivity (0.21), specificity (0.5) and predictive value (0.6). Pooled quarter milk culture analysis had a very low sensitivity (0.17). The kinetics ELISA had lower sensitivity (0.59), specificity (0.90) and predictive value (0.43) as compared to the IS900 PCR assay using pooled quarter milk samples. Results from our study suggest that IS900 PCR assay using bulk tank milk may not be useful for screening herds with M. avium subsp. paratuberculosis infected animals. In conclusion, use of IS900 PCR assay for cows in 2(nd) lactation and higher, using aseptically collected pooled quarter milk samples, can be a useful tool for screening and monitoring lactating cattle in herds with M. avium subsp. paratuberculosis infection.

Animals↗
PubMed2004

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis.

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.

Animals↗
PubMed2005

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays.

AIMS: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. METHODS AND RESULTS: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. CONCLUSIONS: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.

Animals↗
PubMed1992

Detection of Mycobacterium paratuberculosis antigen with colloidal immunogold in naturally infected sheep.

The anti-Mycobacterium paratuberculosis polyclonal serum is proved useful for labelling Mycobacterium paratuberculosis in glutaraldehyde-osmium-fixed and epon-embedded intestinal samples from sheep with clinical symptoms of paratuberculosis. M. paratuberculosis marked with antibody-coated colloidal gold stain was seen in macrophages, epithelioid cells, giant cells and neutrophils throughout intestinal mucosa. In large macrophages with a low lysosomal content, a great number of intact mycobacteria was seen within phagosomes. In macrophages with average lysosomal content, very few intact mycobacteria or mycobacterial debris were present and lysosome-phagosome fusions were observed. Mycobacteria within neutrophils were scanty. These results show the usefulness of colloidal immunogold techniques for studies of the pathogenesis of paratuberculosis.

Animals↗
PubMed2005

Detection and Isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia.

OBJECTIVES: Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johne's disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohn's disease were infected with this pathogen. METHODS: Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing. RESULTS: Twenty five patients (83.3%) with Crohn's disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohn's patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens. CONCLUSIONS: Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohn's disease patients. The finding of the organism colonizing a proportion of people without Crohn's disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

Adolescent↗
PubMed2000

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis.

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.

Animals↗
PubMed2001

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields.

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.

Animals↗
PubMed2005

Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises.

Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants ("7,5") appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.

Animal Husbandry↗
PubMed2004

New latex bead agglutination assay for differential diagnosis of cattle infected with Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis.

Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the "gold standard" culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n = 10), M. avium subsp. paratuberculosis-positive cattle (naturally infected, n = 16; experimentally infected, n = 8), and M. bovis-positive cattle (naturally infected, n = 49;experimentally infected, n = 20) were applied to an EIA and an LBAA developed with ESAT6-p, the two tests showed similar sensitivity (97.1% by EIA, 95.7% by LBAA), high specificity (94.2% by EIA, 100% by LBAA), and a positive correlation (kappa value, 0.85; correlation rate, 93.2%; correlation coefficient, 0.64). Receiver operating characteristic analysis of EIA results and comparison with the culture method determined a suitable cutoff value at 0.469, with an area under the curve of 0.991 (95% confidence interval, 0.977 to 1.0). As LBAA didn't show any positive reactions with sera from uninfected control cows or M. avium subsp. paratuberculosis-infected cattle, which were confirmed to be free of M. bovis by culture or PCR, LBAA using the ESAT6-p can be a rapid and useful M. bovis diagnostic assay. The data suggest that rapid, sensitive, and specific assays can be developed with peptides containing immunodominant epitopes present in proteins uniquely expressed in M. bovis or M. avium subsp. paratuberculosis for differential diagnosis of cattle infected with M. bovis or M. avium subsp. paratuberculosis.

Animals↗
PubMed2001

Use of Hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. paratuberculosis did not significantly alter the number of apoptotic cells.

Animals↗
PubMed1999

Antigen-specific B-cell unresponsiveness induced by chronic Mycobacterium avium subsp. paratuberculosis infection of cattle.

Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne's disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne's disease in cattle.

Animals↗
PubMed2002

Gene expression profiling of peripheral blood mononuclear cells from cattle infected with Mycobacterium paratuberculosis.

A bovine-specific cDNA microarray system containing 721 unique leukocyte expressed sequence tags (ESTs) and amplicons representing known genes was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from clinical and subclinical Johne's disease-positive Holstein cows (n = 2 per group). Stimulation of PBMCs from clinically infected cows with Mycobacterium paratuberculosis tended to decrease expression of 83 genes (fold change, >1.5). Of these 83 genes, 16 displayed significant down regulation across both clinical cows (P < 0.1), including genes encoding microspherule protein 1, fibroblast growth factor, and the Lyn B protein kinase. Only eight genes from PBMCs of clinically infected cows exhibited a modest up regulation following stimulation with M. paratuberculosis, including those encoding bovine CD40L, gamma interferon, interleukin-10 (IL-10), and tissue inhibitor of matrix metalloproteinases (TIMP) 4. In contrast, stimulation of PBMCs from subclinically infected cows with M. paratuberculosis tended to up regulate expression of 71 genes representing 68 unique transcripts. Of these, 11 genes showed significant up regulation (fold change, >1.5; P < 0.1) across both animals, including those encoding bovine CD40L, several matrix metalloproteinases, and SPARC (secreted protein, acidic and rich in cystine). Repression of gene expression was also observed in PBMCs from the subclinical cows, with 16 genes being significantly down regulated (fold change, >1.5; P < 0.1) across both animals, including those encoding the bovine orthologs of cytochrome oxidase subunit III, IL-1 receptor type I, and fibrinogen-like 2 protein. Only one clone, representing an unknown bovine EST, was similarly down regulated in PBMCs from both the clinical and subclinical cows. Thus, the most prominent change induced by exposure of PBMCs from clinical cows to M. paratuberculosis in vitro tended to be repression of gene expression, while changes in similarly treated PBMCs from subclinical cows was balanced between gene activation and repression. Comparison of gene expression profiles between PBMCs from clinical and uninfected (control) cows stimulated with the general mitogen concanavalin A were highly similar (overall r = 0.84), suggesting that M. paratuberculosis-induced gene repression in clinically infected cow PBMCs was not due to a general failure of the immune response in these animals.

Animals↗
PubMed2004

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves.

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.

Animals↗
PubMed2006

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease.

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.

Animals↗
PubMed1993

Cultivation of Mycobacterium paratuberculosis from bovine fecal samples by using elements of the Roche MB Check system.

Components of a commercially available, nonradiometric, biphasic (liquid medium then solid medium) system for the detection of Mycobacterial species, Roche MB Check, were adapted for the isolation of Mycobacterium paratuberculosis from bovine fecal specimens. A two-stage culture procedure was developed in which processed fecal samples were incubated in modified commercial liquid medium and then subcultured onto Herrold's egg yolk medium with mycobactin. By using known culture-positive samples and/or samples from animals clinically affected with paratuberculosis, it was found that visible colonial growth on solid media could be obtained after 4 weeks of incubation in liquid medium containing egg yolk and mycobactin followed by 8 weeks of incubation on Herrold's egg yolk medium. In the second part of the study, conventional fecal culture (sample sedimentation in hexadecylcetylpyridinium chloride followed by incubation on Herrold's egg yolk medium) was compared with the two-stage system using a two-step centrifugation technique for sample preparation. One hundred fecal samples from clinically normal but absorbed-enzyme immunoassay-positive cattle were used for the comparison. Conventional culture yielded a sensitivity of 16.5%, whereas the sensitivity of the two-stage system was 29.4%. When used in parallel, the tests detected 36.5% of the samples. There was no significant difference between the two methods in the time taken to obtain visible colonies. These results indicate that the two-stage method is a sensitive method for isolation of M. paratuberculosis from fecal samples obtained from cattle with clinical paratuberculosis. In addition, the two-stage system is more sensitive than conventional culture for the isolation of M. paratuberculosis from subclinically infected cattle.

Animals↗
PubMed1998

Rapid detection of Mycobacterium paratuberculosis in clinical samples from ruminants and in spiked environmental samples by modified BACTEC 12B radiometric culture and direct confirmation by IS900 PCR.

The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of Mycobacterium paratuberculosis in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of Mycobacterium paratuberculosis by IS900 PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate M. paratuberculosis from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne's disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with M. paratuberculosis. The results suggested that radiometric culture was more sensitive than histopathology in detecting M. paratuberculosis infection in sheep and goats and more sensitive than culture on Herrold's egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of > or = 10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of > or = 10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.

Animals↗
PubMed2005

Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures.

A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrold's Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johne's disease.

Bacterial Typing Techniques↗
PubMed2000

Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.

An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture.

Animals↗