Persistence of Mycobacterium avium subspecies paratuberculosis in endangered Florida Key deer and Key deer habitat.

Mycobacterium avium subsp. paratuberculosis (MAP) was first reported in the endangered Key deer (Odocoileus virginianus clavium) in 1996 on Big Pine Key, Florida, USA. By 2008, eight additional MAP-positive Key deer had been identified on Big Pine Key and the nearby Newfound Harbor Keys. This study was conducted to determine if MAP was still present in Key deer and whether natural or man-made freshwater sources were contaminated with MAP. Between November 2009 and September 2012, MAP was isolated from 36/369 (10%) fecal samples collected from the ground throughout the Key deer range on Big Pine Key and the Newfound Harbor Keys, but all 36 positive samples were from Little Palm Island (36/142 [25%]). Only 1/729 (0.1%) environmental samples was positive; this was from the garden fountain on Little Palm Island (1/81 [1%]). In addition, MAP was detected in 3/43 (7%) necropsied Key deer, all from Little Palm Island (3/3 [100%]). Of these three Key deer, pooled samples from the ileum, cecum, and ileocecal lymph node from two were MAP-culture positive and feces from one of these were culture-positive. The third deer was only PCR-positive. Evidence of MAP was only detected on Little Palm Island during this sampling period and environmental contamination was limited.

Mycobacterium avium subspecies paratuberculosis confirmed following serological surveillance of small ruminants in Grenada, West Indies.

Surveillance for Mycobacterium avium subsp. paratuberculosis (Map) infection in small ruminants of Grenada was undertaken using a commercial enzyme-linked immunosorbent assay (ELISA). Among the 479 sheep tested, 11 (2.3%) were ELISA positive while only 1 out of 260 goats (0.3%) was ELISA positive. Five of the 12 ELISA-positive animals were also positive in a commercial agar gel immunodiffusion (AGID) assay, and 4 of these showed acid-fast rods consistent with Map in fecal smears. Two sheep that were test-positive by ELISA, AGID, and fecal smears were euthanized and necropsied. Both had gross and histological lesions of paratuberculosis affecting the ileocecal area of small intestines and adjacent lymph nodes. These tissues were successfully cultured in 2 of 3 variants of Middlebrook 7H10 medium. The identity of acid-fast organisms isolated from the tissues was confirmed as Map by multiplex conventional polymerase chain reaction. Using IS1311 amplification and Hinf I restriction digest analysis, isolates were identified as cattle (C) strains of Map. The current study describes Map infection in Grenada and confirms the presence of C type in sheep on the island of Carriacou. The low seroprevalence in clinically normal animals on the islands of Grenada and Carriacou suggests that control measures implemented in the near future may have a good chance of preventing spread of the infection.

Paratuberculosis in captive and free-ranging wildlife.

All ruminant species, exotic or domestic, captive or free-ranging, are susceptible to disease and death due to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Young ruminants are the most prone to infection through fecal-oral transmission. Fatal Johne's disease cases have occurred in numerous zoologic hoofstock collections and thus MAP infection is of concern for an industry focused on conserving rare individual animals and their genetics. Diagnosis is best based on MAP detection by PCR or culture in non-domestic species. True nonruminant wildlife reservoirs (ie, a population capable of sustaining the infection independently of reinfection from the initial source and transmitting the pathogen to other species) are rare.

European hares in Chile: a different lagomorph reservoir for Mycobacterium avium subsp. paratuberculosis?

Ruminants are the principal host for infection by Mycobacterium avium subsp. paratuberculosis (Map), the cause of Johne's disease. Based on studies of a Map-infected population of European rabbits (Oryctolagus cuniculus) in Scotland, lagomorphs as a broad taxonomic order were proposed as potential nonruminant reservoirs for Map. To determine whether a different lagomorph species may serve as a wildlife reservoir, we investigated Map infection in European hares (Lepus europaeus) sharing habitat with known Map-infected dairy cattle in southern Chile. Fecal, mesenteric lymph node, and ileal samples were aseptically collected from 385 wild hares for liquid culture and real-time polymerase chain reaction identification of acid-fast isolates. All tissue samples were also acid-fast stained and examined microscopically. We isolated Map from at least one tissue from 48 hares (12.6%) and fecal samples from 16 hares (4.2%). No Map was found in tissues of eight of the fecal-culture-positive hares. Histologically, all tissues from all hares were within normal limits, and no acid-fast organisms were observed in any sample. Active infection, implying amplification of the organism secondary to resultant disease, was not evident. With this report Map isolations on a population versus incidental detection have now been made from two lagomorph species. However, although the rabbit population studied in Scotland appears to function as a Map reservoir, the hares studied in Chile appear to be a dead-end host, serving only as potential mechanical vectors for the organism.

Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts.

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR.

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

Surveys for disease agents in introduced elk in Arkansas and Kentucky.

Surveys for disease agents were conducted in introduced free-ranging elk (Cervus elaphus nelsoni) in Arkansas and Kentucky. Elk had been captured in Colorado and Nebraska and released in Arkansas during 1981-1985. From 1997 through 2002 elk were captured in Arizona, Kansas, North Dakota, New Mexico, Oregon, and Utah and released in southeastern Kentucky. Specimens were collected from 170 hunter-killed elk in Arkansas during 1998-2006, and 44 elk in Kentucky during 2001-2004. Significant findings included isolation of Mycobacterium avium paratuberculosis from one elk in Kentucky and evidence of previous or current infections by Parelaphostrongylus tenuis in several animals in Arkansas. Serological tests provided evidence of previous infection by epizootic hemorrhagic disease virus, bluetongue virus, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza-3 virus, and multiple serovars of Leptospira interrogans. Mycobacterium bovis, Brucella abortus, chronic wasting disease (CWD), and hemoparasites such as Anaplasma spp. were not detected. Results from elk obtained through these surveys were consistent with exposure to disease agents endemic in livestock and wildlife in Arkansas and Kentucky.

Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients.

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P<0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.

Production of and applications for a polyclonal IgY diagnostic reagent specific for Mycobacterium avium subsp. paratuberculosis.

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-la-beled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immunomagnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.

Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis.

OBJECTIVES: Mycobacterium avium subspecies paratuberculosis (MAP) has been targeted for treatment with clarithromycin and rifamycin derivatives in numerous cases of Crohn's disease (CD). 6-Mercaptopurine and its pro-drug azathioprine are widely used as immunomodulators in the treatment of CD and have recently been shown to have anti-MAP activity in vitro. The objectives of the study were to evaluate the in vitro effects on MAP of (i) 6-mercaptopurine when combined with each of eight conventional antibacterial agents with in vitro anti-MAP activity and (ii) antibacterial combinations consisting of two drugs (clarithromycin combined with amikacin, rifampicin, ciprofloxacin or ethambutol) and three drugs (clarithromycin, rifabutin and clofazimine). METHODS: The drug interaction effects on nine human isolates of MAP were determined by the chequerboard method adapted for the BACTECMGIT960 culture system and by calculation of the fractional inhibitory concentration index (FICI) for drug combinations. RESULTS: Synergism (FICI < or = 0.5) was observed between 6-mercaptopurine and azithromycin (seven isolates), clarithromycin, rifampicin, rifabutin (four isolates each) and ethambutol (two isolates). 6-Mercaptopurine was not antagonistic with any of the antibacterial agents tested. Among the combinations of two and three antibacterials tested, the clarithromycin/rifampicin combination was synergistic against four isolates, while all other combinations showed no interaction. CONCLUSIONS: This in vitro study suggests that 6-mercaptopurine may be synergistic with macrolides and rifamycin derivatives against MAP. The activity of clarithromycin against MAP seems to be enhanced by rifampicin.

Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs.

OBJECTIVES: To evaluate the BACTEC(TM) MGIT(TM) 960/MGIT Para TB (MGIT) system for drug susceptibility testing of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some forms of Crohn's disease. METHODS: MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, the BACTEC(TM)460TB system (BACTEC) and conventional agar dilution methods. RESULTS: MICs determined by MGIT methods showed 80%-100% agreement (+/-1 log(2) dilution) with those determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromycin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+/-1 log(2) dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at 2 log(2) dilution differences. For clarithromycin, the MGIT method had greater agreement with the agar dilution method (70% at the same dilution) than the BACTEC method (60% at +/-1 log(2) dilution); agreement increased to 100% at +/-2 log(2) dilutions in both cases. The MGIT and agar dilution methods agreed 60% and 100% for amikacin MICs at +/-1 log(2) dilution and +/-2 log(2) dilutions, respectively. By all methods MICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100% agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80% agreement for rifampicin using published MIC thresholds available for M. avium complex strains. CONCLUSIONS: This study shows that the MGIT system can be used for rapid and reliable drug susceptibility testing of MAP.

Rapid mycobacterial liquid culture-screening method for Mycobacterium avium complex based on secreted antigen-capture enzyme-linked immunosorbent assay.

Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.

Johne's disease in a free-ranging white-tailed deer from Virginia and subsequent surveillance for Mycobacterium avium subspecies paratuberculosis.

Johne's disease (paratuberculosis) was diagnosed in a 2-yr-old, male, free-ranging white-tailed deer (Odocoileus virginianus) from Fauquier County, Virginia, USA, based on histopathology and culture for Mycobacterium avium subspecies paratuberculosis. Clinical and pathologic findings included emaciation; loss of body fat; chronic diarrhea; severe, chronic, diffuse granulomatous colitis with intrahistiocytic acid-fast bacilli; moderate, chronic granulomatous lymphadenitis with intrahistiocytic acid-fast bacilli; as well as moderate chronic, multifocal, lymphoplasmacytic hepatitis. These findings are consistent with previous reports of Johne's disease in cervids. Subsequent targeted surveillance of 10 emaciated deer with diarrhea, as well as sampling of 72 asymptomatic deer for M. avium subsp. paratuberculosis using culture of multiple tissue types, as well as serology using an enzyme-linked immunosorbent assay (ELISA) optimized for cervid antibody detection, did not reveal any additional cases of infection in this geographic region. To date, this appears to be an isolated case of Johne's disease in a free-ranging white-tailed deer, and infection with the causative agent for Johne's disease appears to be an infrequent occurrence in deer from this region. The origin of infection was most likely domestic ruminants. This is the first report of clinical Johne's disease in a free-ranging white-tailed deer outside of the Florida Keys, USA. Stressors, such as high deer population density and low selenium levels, may have contributed to the development of clinical disease in this case and warrant further investigation.

Distribution of Mycobacterium avium subspecies paratuberculosis in the lower Florida Keys.

Johne's disease, a fatal and contagious gastrointestinal infection caused by Mycobacterium avium subsp. paratuberculosis (Map), was first diagnosed in an endangered Florida Key deer (Odocoileus virginianus clavium) in 1996 and later in six additional Key deer deaths from 1998 to 2004. We investigated the geographic distribution of Map in the Lower Florida Keys from February 2005 through May 2006 via collection of blood and fecal pellets from 51 live-captured deer, collection of 550 fecal samples from the ground, and by necropsies of 90 carcasses. Tissue and fecal samples also were submitted from 30 raccoons (Procyon lotor), three feral cats (Felis catus), an opossum (Didelphis virginiana), and a Lower Keys marsh rabbit (Sylvilagus palustris hefneri). Mycobacterium avium subsp. paratuberculosis was identified in 23 Key deer fecal samples collected from the ground, tissue samples from two clinically ill Key deer, and from the mesenteric lymph node of a raccoon. The results of this study indicate that Map persists in the Key deer population and environment at a low prevalence, but its distribution currently is limited to a relatively small geographic area within the range of Key deer.

Familial associations with paratuberculosis ELISA results in Texas Longhorn cattle.

The objective of this cross-sectional study was to estimate familial associations with paratuberculosis ELISA status in beef cattle. Texas Longhorn cattle (n=715) greater than 2years of age were sampled for paratuberculosis testing using ELISA and fecal culture. Diagnostic test results were indicative of substantial numbers of false-positive serological reactions consistent with environmental exposure to non-MAP Mycobacterium spp. Associations between ancestors and paratuberculosis ELISA status of offspring were assessed using conditional logistic regression. The association between ELISA status of the dam and her offspring was assessed using linear mixed-effect models. Significant associations were identified between some ancestors and offspring ELISA status. The odds of being classified as "suspect" or greater based on ELISA results were 4.6 times greater for offspring of dams with similarly increased S:P ratios. A significant positive linear association was also observed between dam and offspring log-transformed S:P ratios. Results indicate that there is familial aggregation of paratuberculosis ELISA results in beef cattle and suggest that genetic selection based on paratuberculosis ELISA status may decrease seroprevalence. However, genetic selection may have minimal effect on paratuberculosis control in herds with exposure to non-MAP Mycobacterium spp.

Environmental mycobacteria in soil and water on beef ranches: association between presence of cultivable mycobacteria and soil and water physicochemical characteristics.

Exposure to environmental mycobacteria has been reported to be a factor contributing to false-positive results on bovine serological tests detecting antibodies to Mycobacterium avium subsp. paratuberculosis (Mptb). This study was conducted to investigate the association between recovery of mycobacteria from the environment of cattle and both (i) historically high or low seroprevalence to Mptb, and (ii) soil and water physicochemical characteristics. Eighty-two samples (soil and water) from nine beef cattle ranches in South-central and South Texas were assessed for the presence of mycobacteria. Twelve mycobacterial species were cultured from soil and water from four herds; no Mptb were detected in environmental samples. A positive culture of environmental mycobacteria from soil was significantly associated with lower pH and calcium as well as higher iron, zinc and manganese contents. Beef cattle are likely to be exposed to environmental mycobacteria that may contribute to false-positive results on ELISAs for Mptb infection. Exposure rates to these mycobacteria likely vary across small geographical areas and may be related to soil and/or water physicochemistry.

Impact of Corynebacterium pseudotuberculosis infection on serologic surveillance for Johne's disease in goats.

False-positive results on serologic assays for Mycobacterium avium subsp. paratuberculosis (MAP) are believed to occur due to cross-reacting antibody produced by Corynebacterium pseudotuberculosis (C. pstb) infection in goats. This issue of compromised specificity was evaluated by testing 771 adult goats from 10 Midwestern goat herds in 2004. Assays for MAP infection included radiometric fecal culture and 2 enzyme-linked immunosorbent assays (ELISAs); ELISA-positive samples were tested by agar gel immunodiffusion (AGID). A synergistic hemolysin inhibition assay (SHI) was used to detect C. pstb antibody. Four infection status categories were evaluated. Category 1 goats (free of both MAP and C. pstb infection) tested negative on all MAP fecal cultures and SHI tests. Five of 181 goats were positive in both ELISAs, and 2 more were positive in ELISA-1 only. For Category 2 (MAP infected; no C. pstb infection), all animals were SHI negative. Six goats were fecal culture positive and strongly positive in both ELISAs; 2 more goats were positive only in ELISA-1. For Category 3 (C. pstb infected or vaccinated; no history of MAP infection), all fecal cultures were negative and 91% were SHI test-positive. In this population, only 2 goats were positive in both MAP ELISAs, while 84 additional goats were test-positive only on ELISA-1. In the absence of C. pstb infection, both ELISAs performed comparably, but when C. pstb infection was present the performance of ELISA-1 was significantly perturbed. Use of the ELISA-2 for goats is an effective and efficient method for Johne's disease surveillance in any goat herd.

Rapid and reliable method for quantification of Mycobacterium paratuberculosis by use of the BACTEC MGIT 960 system.

A simple method for the enumeration of viable Mycobacterium paratuberculosis cells was developed and evaluated using the MGIT 960 culture system. For each of 12 M. paratuberculosis strains isolated from either cattle or humans, single-cell suspensions of M. paratuberculosis cells were adjusted to an optical density at 600 nm of 1.00 (10(7.6) to 10(8.2) cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log10 CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (R2 of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log(10) estimations using the universal standard curve, each subgroup's standard curve, and strain-specific curves were +/-0.87, +/-0.45, and +/-0.31 log10 units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the M. paratuberculosis strain or inoculum volume. In comparison with the previously described BACTEC 460 M. paratuberculosis counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable M. paratuberculosis cells, such as drug susceptibility testing or environmental survival studies.

Association of fecal shedding of mycobacteria with high ELISA-determined seroprevalence for paratuberculosis in beef herds.

OBJECTIVE: To evaluate the seroprevalence of paratuberculosis by use of 2 commercial ELISAs in association with prevalence of fecal shedding of mycobacteria within beef cattle herds. DESIGN: Cross-sectional field study. ANIMALS: Six beef herds (affected herds; 522 cattle) with and 3 geographically matched herds (181 cattle) without high seroprevalence of paratuberculosis. PROCEDURES: Blood and fecal samples were collected from adult cattle and assessed for serum anti-Mycobacterium avium subsp paratuberculosis (MAP) antibodies with 2 commercial ELISA kits and submitted for bacterial culture for MAP and environmental bacteria (termed environmental mycobacteria) via a radiometric method, respectively. Species of mycobacterial isolates were identified, and sensitivities and specificities of the 2 ELISAs were compared. RESULTS: Compared with comparison cattle, cattle from affected herds were 9.4 times as likely to have environmental mycobacteria isolated from feces. Among the 6 affected and 3 comparison herds, the proportions of cattle shedding environmental mycobacteria were 0.225 (range, 0.1 to 0.72) and 0.04 (range, 0 to 0.06), respectively. Although relative MAP- detection specificities (compared with bacterial culture of feces) were different between the 2 ELISAs, sensitivities were not. Nine environmental mycobacterial species were identified from participating herds. All affected herds apparently had > or = 1 bovid infected with MAP, although MAP was not isolated from any cattle in comparison herds. CONCLUSIONS AND CLINICAL RELEVANCE: In beef herds with persistently high rates of false- positive ELISA results, which may be associated with recovery of environmental myco- bacteria from feces, organism detection via bacterial culture of feces or PCR assay should direct paratuberculosis control measures.