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.

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.

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.

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.

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.

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.

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.

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.

Contribution of environmental mycobacteria to false-positive serum ELISA results for paratuberculosis.

OBJECTIVE: To evaluate the effect of exposure to environmental mycobacteria on results of 2 commercial ELISAs for paratuberculosis in cattle. DESIGN: Experimental trial. ANIMALS: 19 weaned crossbred beef calves. PROCEDURES: Calves were inoculated SC with 1 of 5 mycobacterial isolates (3 calves/isolate) derived from herds with high proportions of false-positive serologic reactions for paratuberculosis, Mycobacterium avium subsp paratuberculosis (MAP; positive control inoculum; 2 calves), or mineral oil (negative control inoculum; 2 calves). Sera were assessed at intervals by use of 2 ELISAs (A and B) for paratuberculosis in cattle, and all calves underwent tuberculosis testing at the end of the study. RESULTS: Neither mineral oil-inoculated calf had positive results with either ELISA during the study. Both MAP-inoculated calves were identified as seropositive via ELISA-A, and 1 calf was identified as seropositive via ELISA-B. By use of ELISA-A, > or = 1 false-positive reaction over time was detected in 2, 3, 3, and 1 of the 3 calves injected with Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, or Mycobacterium terrae, respectively. By use of ELISA-B, only M scrofulaceum induced false-positive reactions (2/3 calves). Calves that had at least 1 positive ELISA-A result were more likely to be classified as suspect reactors via the caudal fold tuberculosis test. CONCLUSIONS AND CLINICAL RELEVANCE: False-positive serologic reactions may occur during use of commercially available ELISAs for paratuberculosis in calves experimentally exposed to environmental mycobacteria; naturally occurring exposures with these mycobacteria may represent a cause for high proportions of false-positive serologic reactions for paratuberculosis in some cattle herds.

Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp. avium infections in a tule elk (Cervus elaphus nannodes) herd.

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P) > or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P > or = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.

Performance of a Johne's disease enzyme-linked immunosorbent assay adapted for milk samples from goats.

Antibody detection-based tests for paratuberculosis offer speed and economy, 2 diagnostic test attributes important to animal industries with narrow profit margins. Application of such tests to individual milk samples instead of serum samples can further improve testing efficiency and decrease testing cost. Accuracy of a commercial bovine paratuberculosis enzyme-linked immunosorbent assay (ELISA) adapted for use on goat serum and milk samples was determined. Fecal, blood, and milk samples were collected from 159 goats belonging to 2 Wisconsin goat herds with a prior history of paratuberculosis and 1 herd of 50 goats from a paratuberculosis-free Wisconsin herd. Fecal samples were cultured using the modified BACTEC 12B media. Sera were tested according to the manufacturer's instructions for bovine samples. Milk samples were centrifuged and mixed with the ELISA kit's Mycobacterium phlei-containing diluent at a ratio of 1:2. Using fecal culture as the "gold standard," the sensitivity of the ELISA on goat serum was 64% and the sensitivity of the ELISA on goat milk was 48%. The milk ELISA had higher agreement with fecal culture results (kappa = 0.525) than the serum ELISA (kappa = 0.425). ELISA specificity was 100% on both serum and milk. Regression analysis also showed good correlation between serum and milk S/P values (r2 = 0.67). Although less sensitive, the ELISA on goat milk samples appears to offer a useful, low-cost alternative for detection of goats with paratuberculosis that have progressed to the stage of shedding M. paratuberculosis in their feces.

Protein G binding to enriched serum immunoglobulin from nondomestic hoofstock species.

Quick and cost-effective serologic assays, such as those based on enzyme-linked immunosorbent assay (ELISA) technology, are useful for screening animal populations for infectious diseases. Recombinant protein G is described as an almost universal ELISA conjugate for the detection of antibodies from a wide range of animal species. However, there is limited data documenting the ability of protein G to bind immunoglobulin (Ig) from many captive and free-ranging nondomestic hoofstock (Order Artiodactyla, e.g., elk, antelope, bison). Protein G binding to Ig from 11 species within this taxonomic order (addax, antelope, bison, bontebok, elk, impala, kudu/nyala, muntjac, oryx, sheep, and white-tailed deer) and 2 control species (bovine and chicken) was assessed. A serum Ig enrichment protocol, using high-performance liquid chromatography (HPLC), was optimized in bovids (Bos taurus) and then applied to the other study species. Binding assays were performed by adding protein G to microtiter wells coated with titrated dilutions of enriched artiodactyl Ig. Optical densities were measured and binding curves generated. Differences in protein G binding were observed, both within and among species, as well as within taxonomic families. Significant intraspecies binding variation was observed for 7 species tested (antelope, oryx, sheep, muntjac, impala, bontebok, and addax). No statistically significant intraspecies differences in protein G binding were found for Ig from bison, elk, kudu/nyala, white-tailed deer, plus control species (cattle and chicken). Binding of protein G to Ig from impala, muntjac, and elk was statistically different from the positive control (cattle), with muntjac binding curves statistically comparable with the negative control (chicken). For the other 7 species tested, binding curves illustrated the ability of protein G to bind Ig as well as, or better than, the positive control. These findings expand the list of animal species whose Ig is capable of being detected using recombinant protein G, with the caveat that protein G does not bind Ig uniformly in closely related species. It is concluded that recombinant protein G conjugates may serve as useful reagents for serodiagnosis by ELISA in nondomestic hoofstock, although different assay interpretation algorithms and assay protocols may need to be developed on a per species basis for maximum diagnostic effectiveness.

Diagnostic testing patterns of natural Mycobacterium paratuberculosis infection in pygmy goats.

Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFNgamma), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFNgamma response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNgamma results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats.

Culture and serologic survey for Mycobacterium avium subsp. paratuberculosis infection among southeastern white-tailed deer (Odocoileus virginianus).

From July 1998 through October 2002, radiometric culture (ileocecal lymph node, mesenteric lymph node, and feces) and serologic testing by enzyme-linked immunosorbent assay (ELISA) were used to survey white-tailed deer (Odocoilens virgianus) from the soutlheastern United States for infection by Mycobacterium avium subsp. paratuberculosis (Mptb), the causative agent of paratuberculosis (Johne's disease). Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one of 313 deer (0.3%) originating from 63 populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Six deer (2%), all from different populations, had ELISA results above a 0.25 sample-to-positive cutoff value, but none of the ELISA reactors originated from the population from which the single Mptb isolation was made. These six deer were seronegative when tested by agar gel immunodiffusion (AGID). Collectively, these data indicate that white-tailed deer currently do not constitute a broad regional reservoir for Mptb; however, further study is warranted to clarify the significance, if any, of infected deer to the epizootiology of paratuberculosis on a local scale. Adaptation and validation of an ELISA or another serologic assay for use with deer and other wildlife would markedly enhance Mptb surveillanece among wild populations and would be a powerful tool for gaining information on the role of wild species in epidemiology of paratuberculosis.

Paratuberculosis in key deer (Odocoileus virginianus clavium).

Paratuberculosis was diagnosed in an endangered Key deer (Odocoileus virginianus clavium) in November 1996. Between 10 April 1997 and 28 September 2000, the Key deer population was monitored for infection with Mycobacterium avium subsp. paratuberculosis by necropsy of available carcasses (n = 170), fecal cultures, and serology. One additional clinically affected Key deer was discovered in July 1998, and M. avium subsp. paratuberculosis was cultured from the feces of one live, asymptomatic deer. The results of this study provided sufficient evidence to consider the Key deer herd infected with M. avium subsp. paratuberculosis at very low prevalence.

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.