Evaluation of environmental sampling and culture to determine Mycobacterium avium subspecies paratuberculosis distribution and herd infection status on US dairy operations.

The objectives of this study were to determine the distribution of Mycobacterium avium subspecies paratuberculosis (MAP) in the environment and assess the relationship between the culture status of MAP in the farm environment and herd infection status. The National Animal Health Monitoring System's Dairy 2002 study surveyed dairy operations in 21 states. One component of the study involved collection and culturing of environmental samples for MAP from areas on farms where manure accumulated from a majority of a herd's cows. Operations were selected for inclusion based on perceived risk factors for MAP infection identified in a previously administered questionnaire. Individual animal and environmental samples were collected and used to determine the efficiency of environmental sampling for determination of herd infection status. Individual animal fecal, serum, and milk samples were used to classify herds as infected or not infected based on the presence of at least one test-positive animal in the herd. A total of 483 environmental samples (approximately 5 per farm) were collected, and 218 (45.1%) were culture-positive for MAP. A similar percentage of environmental cultures collected from all designated areas were positive [parlor exits (52.3%), floors of holding pens (49.1%), common alleyways (48.8%), lagoons (47.4%), manure spreaders (42.3%), and manure pits (41.5%)]. Of the 98 operations tested with the environmental sample culture, 97 had individual serum ELISA results, 60 had individual fecal culture results, and 34 had individual milk ELISA results. Sixty-nine of the 98 operations (70.4%) had at least one environmental sample that was culture-positive. Of the 50 herds classified as infected by fecal culture, 38 (76.0%) were identified by environmental culture. Two of the 10 operations classified as not infected based on individual animal fecal culture were environmental culture-positive. Of the 80 operations classified as infected based on serum ELISA-positive results, 61 (76.3%) were identified as environmental-positive, whereas 20 of the 28 (71.4%) operations identified as infected based on milk ELISA were detected by environmental sampling. Environmental sample culturing is less costly than individual animal sampling, does not require animal restraint, and identified more than 70% of infected operations. Environmental sampling is another diagnostic tool that veterinarians and dairy producers can use to determine herd infection status for MAP.

Fatal mycobacteriosis with hepatosplenomegaly in a young dog due to Mycobacterium avium.

Cases of disseminated Mycobacterium avium infections in dogs are rare because it appears that the species is innately resistant to infection. A 2-year-old, castrated, 5 kg Shih Tzu-Poodle-cross developed anemia, abdominal pain, lethargy, and splenomegaly. Histological examination of surgically removed spleen indicated marked granulomatous splenitis with myriad intracytoplasmic acid-fast bacterial rods. Ultrastructural examination revealed the presence of 3-4-microm-long mycobacteria in phagolysosomes of epithelioid macrophages. Tissue extract of lightly fixed spleen was positive for M. avium 16S ribosomal RNA and negative for M. tuberculosis complex IS6110 DNA by polymerase chain reaction testing. Anemia was associated with the presence of mycobacteria-infected macrophages in bone marrow. The animal's condition deteriorated, and euthanasia was performed after a clinical course of 2 months. The principal morphological findings at necropsy were severe diffuse granulomatous hepatitis, enteric lymphadenomegaly, and segmental granulomatous enteritis with intralesional mycobacteria present. Mycobacterium avium was cultured from enteric lymph nodes sampled at necropsy. The source of infection was not established but was presumed to be environmental with an enteric portal of entry.

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain.

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.

Identification of Brucella abortus strain 19 by decreased ability to utilize erythritol as determined by gas liquid chromatography.

A method to identify Brucella abortus strain 19 by erythritol utilization using gas liquid chromatography (GLC) was developed. A total of 69 strains of B. abortus (41 virulent field strain isolates and 28 strain 19 isolates) were tested. Following incubation of the isolate with a standard amount of erythritol, the erythritol present in the cell suspension was acetylated and measured by GLC. Field strains of B. abortus utilized an average of 90.9% of the erythritol, whereas vaccine strains utilized an average of 42.4%. This difference in erythritol utilization will allow a more rapid identification of B. abortus strain 19.

Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus).

A culture isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) was characterized. The isolate was a gram-negative coccobacillus, and the colonial morphology was typical of a smooth Brucella. The isolate was positive for catalase, oxidase, nitrate reduction, and urease. Hydrogen sulfide was not produced. It grew in air at 37 C but required 72 hours for good growth. There was growth on media containing basic fuchsin, thionin, thionin blue, penicillin, and erythritol. The M antigen was dominant, and the isolate was lysed by 4 of 10 brucellaphages tested. The oxidative metabolic profile of the isolate was similar to that for B. abortus but differed in utilization of L-asparagine, L-glutamic acid, and DL-citrulline. Whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles were markedly different from the protein profiles of reference strains of Brucella species. Biochemical and oxidative metabolism profiles indicated that the isolate belongs in the genus Brucella but did not match the profiles of any established species or biovars. This isolate may be an atypical strain of a recognized Brucella species or a new biovar or species of Brucella.

Restriction fragment length polymorphism analysis of Mycobacterium bovis isolates from captive and free-ranging animals.

Mycobacterium bovis isolates from cattle, captive elk, and free-ranging mule deer and coyotes were examined by restriction fragment length polymorphism (RFLP) analysis. DNA extracted from each isolate was digested with restriction endonucleases AluI and PvuII. DNA probes used for Southern hybridizations were a 37-base oligonucleotide and a 123-base-pair sequence specific for the insertion sequence IS6110 and a plasmid, pTBN12, which contains a polymorphic GC-rich repetitive sequence present in several species of mycobacteria. Generally, M. bovis isolates originating from a single herd of either cattle or captive elk had identical RFLP patterns, whereas isolates from unrelated sources had distinct patterns. The RFLP patterns for M. bovis isolates from free-ranging mule deer and coyotes were identical to patterns observed for isolates from a captive elk herd that was located in the area where the free-ranging animals were found. These results indicate that the captive elk herd may have been the source of M. bovis that infected the free-ranging animals. Results of this study show that RFLP analysis is a useful tool for differentiation of M. bovis isolates and for molecular epidemiology studies to determine possible sources of infection in outbreaks of tuberculosis in animals.

Comparison of postmortem techniques for the detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus).

A retrospective study of various diagnostic postmortem techniques used in a 4-year surveillance program for detection of Mycobacterium bovis infection in wild white-tailed deer (Odocoileus virginianus) was conducted. The tests evaluated were routine histopathology, acid-fast staining, detection of acid-fast bacilli in culture, and an M. tuberculosis group-specific genetic probe applied to pure cultures. Each of these techniques were compared with a reference or "gold standard" of mycobacterial culture and identification. Histopathology, the most rapid form of testing for M. bovis infection in white-tailed deer samples, had a sensitivity of 98% and a specificity of 87%, resulting in a positive predictive value of 94%. The detection of acid-fast bacilli by staining was less sensitive than histopathology (90%), but its higher specificity (97%) resulted in a positive predictive value of 99%. The detection of acid-fast bacilli on culture was both highly specific (93%) and sensitive (100%). The group-specific genetic probe had the highest sensitivity and specificity and produced results in complete agreement with those of mycobacterial culture, suggesting that this technique could be used as the new "gold standard" for this particular wildlife tuberculosis surveillance program.

Effect of environmental-genetic interactions on Mycobacterium avium challenge infection.

Chickens from lines selectively bred for either a high-antibody (HA) or low-antibody (LA) response to sheep erythrocytes were injected intravenously with Mycobacterium avium while being held in low, medium, or high levels of social stress for 5 days (first environment). During the remaining 6 weeks, they were held under either low or medium levels of social stress (second environment). Infection led to lesions consisting of granulomas, some of which had necrotic centers. There was a positive correlation between numbers of lesions with necrotic centers and M. avium cells recovered from livers. The numbers and nature of lesions were influenced by both genetic and environmental factors. Numbers of necrotizing lesions increased with stressfulness of the first environment. Total numbers of lesions were reduced by the medium-stress second environment, and the total number of necrotizing lesions was reduced among LA chickens in the low-stress second environment.

Characteristics of Salmonella spp. and Escherichia coli isolated from broiler flocks classified as "good" or "poor" producers.

Cecal samples from 100 broiler flocks were cultured for Escherichia coli and Salmonella. Samples were selected from flocks classified as either "good" or "poor" producers by a production formula. In an attempt to identify predictors of flock productivity, isolates were studied for differences in antibiotic resistances, hemagglutination of erythrocytes, production of colicins, production of siderophores, type of hemolysis, resistance to host complement, and presence of plasmids. S. typhimurium (copenhagen) was isolated from one poor producing flock and three good producers. Salmonella isolates showed no significant differences in the parameters studied. The E. coli isolates showed significant differences only for the presence of plasmids. These data indicate that differences in host intestinal E. coli from good and poor producing flocks do not predict flock productivity.

A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus).

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph node (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84.97%) and 88% [CL 77.94%], respectively, and the specificity of both was 89% [CL 85.92%]). The sensitivity and specificity of histopathology II were 89% (CL 79.95%) and 77% (CL 72.81%), respectively. The highest sensitivity estimates (93-95% [CL 84.98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%] were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results showed that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.

Comparison of phenotypic characteristics of Salmonella spp isolated from healthy and ill (infected) chickens.

Phenotypic characteristics of 12 paired, Salmonella serotypes isolated from healthy and ill chickens were compared. Variables compared included antibiotic resistance profiles, production of colicins and siderophores, mannose-sensitive hemagglutination of erythrocytes, resistance to serum complement, carbon source utilization, presence and transmissibility of R plasmids, and invasiveness in primary chicken kidney cell culture. Differences were found between pairs for utilization of carbon sources, mannose-sensitive hemagglutination of erythrocytes, and invasiveness in cell culture.

Application of pulsed-field gel electrophoresis for differentiation of vaccine strain RB51 from field isolates of Brucella abortus from cattle, bison, and elk.

Restriction endonuclease patterns of genomic fragments separated by use of pulsed-field gel electrophoresis were used to differentiate Brucella abortus strain RB51, a rifampin-resistant mutant of the standard virulent strain 2308, from other brucellae. Results were compared with results obtained by use of standard methods for characterizing brucellae. Electrophoretic patterns of the ATCC type strains allowed identification of the strains to the level of species. Genomic profiles of B abortus biovars 1, 2, and 4 were similar, as were those of biovars 5, 6, and of biovar 3 was similar to that of biovars 5, 6, and 9, except for a missing band at 93 kb and additional bands at 65 and 67 kb. A different fingerprint was detected in B abortus strain RB51, using the pulsed-field gel electrophoresis patterns of genomic DNA digested with restrictive endonuclease Xba I. The profile of B abortus strain RB51 contained a band at 104 kb, as opposed to a 109-kb fragment within profiles of B abortus isolates from naturally infected cattle, bison, and elk. Despite known biochemical and biological differences between RB51 and its parent strain (2308), restriction endonuclease analysis results were similar.

Identification of tuberculosis in cattle slaughtered in Mexico.

OBJECTIVES: To determine epidemiologic factors associated with tuberculosis (TB) in dairy cattle slaughtered in 6 important regions for milk production in Mexico. ANIMALS: 2,500 cattle. PROCEDURE: Tissue specimens with lesions typical of TB were obtained during routine inspection of carcasses at abbatoirs between July 1996 and January 1997. Infection with Mycobacterium organisms was confirmed by histologic examination and bacteriologic culture. Species identification was made by use of selective growth medium, conventional biochemical tests, and radiometric procedures. Epidemiologic information for affected cattle was obtained by personal interviews with cattle dealers and owners. RESULTS: 400 (16%) of 2,500 cattle carcasses had gross lesions typical of TB. Of the 400 infected cattle, 336 (84%) had lesions in > or = 1 lymph node. Infection was confirmed in 87% of cattle with gross lesions by histologic examination, in 77% by bacteriologic culture at a laboratory in the United States, and in 59% by bacteriologic culture at a laboratory in Mexico. Most cattle were adult females in fair to good body condition that came from large herds (> 500 cattle) and were not included in the Mexican TB control program. CONCLUSIONS AND CLINICAL RELEVANCE: Mean prevalence of lesions typical of TB in dairy cattle at 6 locations in Mexico was 16%. Mycobacterium infection was confirmed by various techniques in most lesions. Recognition of typical gross lesions at slaughter may expedite TB control procedures.

Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico.

OBJECTIVE: To assess phylogenetic relationships among Mycobacterium bovis isolates by use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting and to relate genetic profiles of isolates to epidemiologic characteristics. ANIMALS: 400 cattle with tuberculosis. PROCEDURE: Mycobacterium bovis was isolated from various organs of cattle slaughtered in 6 geographic regions of Mexico. Most cattle were adult Holsteins from large herds that did not participate in a tuberculosis control program. Four random primers and 2 selected primers were used in RAPD-PCR fingerprinting of 88 isolates. Pairwise genetic distance between isolates was obtained and subjected to cluster analysis with bootstrapping to test for levels of support. RESULTS: 98 different fragments were obtained; there was broad genetic diversity among isolates, and each isolate had a unique RAPD-genotype, including those originating from the same herd. Clustering by geographic location, affected organ, or severity of lesion was not detected. Linkage disequilibrium analysis suggested that M. bovis was highly clonal and that mutations develop at a rapid rate among isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Use of RAPD-PCR could not differentiate M. bovis isolates by epidemiologic characteristics or identify common sources of infection.

Comparison of the sensitivity of the caudal fold skin test and a commercial gamma-interferon assay for diagnosis of bovine tuberculosis.

A study to determine and compare the sensitivity of the caudal fold tuberculin test (CFT) and a commercial gamma-interferon (gamma-IFN) assay for diagnosis of bovine tuberculosis was conducted. A dairy herd with approximately a third of the cattle infected with Mycobacterium bovis was chosen for this study. All cattle from this herd were slaughtered, and tissue specimens for bacteriologic culturing and histologic examination were collected. Results of the CFT and gamma-IFN assay were compared with results of bacteriologic culturing and histologic examination to determine test sensitivity. Results were analyzed, using each of the following 4 standards to classify cattle as infected: positive test result by bacteriologic culturing only; histologic examination only; bacteriologic culturing and histologic examination; and bacteriologic culturing or histologic examination. Sensitivity of the CFT ranged from 80.4 to 84.4%, depending on the standard of comparison. Sensitivity of the gamma-IFN assay ranged from 55.4 to 97.1%, depending on the standard of comparison and on the method of interpretation. The CFT was significantly (P < 0.001) more sensitive than the gamma-IFN assay for diagnosis of bovine tuberculosis when the gamma-IFN assay was conducted and interpreted as instructed by the manufacturer. Maximum overall sensitivity was achieved when results of the CFT and gamma-IFN assay were interpreted in parallel.

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis.

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.

Bovine tuberculosis in a free-ranging mule deer (Odocoileus hemionus) from Montana.

A survey of 41 mule deer (Odocoileus hemionus) and three white-tailed deer (O. virginianus) for bovine tuberculosis was conducted on a Montana (USA) cattle ranch from 2 November 1993 through January 1994. Gross and microscopic lesions typical of tuberculosis were present in tonsil and lymph nodes of the head, thorax, and abdomen of one adult female mule deer. Additionally, a single microgranuloma considered morphologically suggestive of tuberculosis was present in one lymph node of the head of a second mule deer. Mycobacterial isolates from lymph nodes of the head and thorax of the first deer were identified as Mycobacterium bovis.

Abortion caused by Brucella abortus biovar 1 in a free-ranging bison (Bison bison) from Yellowstone National Park.

A near-term aborted bison (Bison bison) fetus was collected near Old Faithful geyser in Yellowstone National Park, Wyoming (USA). On necropsy, the fetus liver had a small capsular tear, and there was a small quantity of blood in the peritoneal cavity. Microscopic lesions included mild, purulent bronchopneumonia and mild, multifocal, interstitial pneumonia. Brucella abortus biovar 1 was isolated from fetal abomasal contents, lung, and heart blood.

Mycobacterium bovis in coyotes from Michigan.

During a survey for tuberculosis in wild carnivores and omnivores, Mycobacterium bovis was cultured from pooled lymph nodes of three adult female coyotes (Canis latrans) harvested by hunters in Michigan (USA). No gross or histologic lesions suggestive of tuberculosis were seen in these animals. One coyote was taken from Montmorency county and two coyotes from Alcona county located in the north-eastern portion of Michigan's Lower Peninsula where free-ranging white-tailed deer (Odocoileus virginianus) have been found infected with bovine tuberculosis. It is thought that these coyotes became infected with M. bovis through the consumption of tuberculous deer. Other species included in the survey were the opossum (Didelphis virginiana), raccoon (Procyon lotor), red fox (Vulpes vulpes), bobcat (Felis rufus), and badger (Taxidea taxus).

Survey of free-ranging elk from Wyoming and Montana for selected pathogens.

From December 1991 through January 1995, a disease survey was conducted on herds of free-ranging, hunter-killed elk (Cervus elaphus nelsoni) from three areas in proximity to Yellowstone National Park (YNP), Wyoming (USA), after tuberculosis caused by Mycobacterium bovis was discovered in a captive herd of elk in the area. Complete or partial sets of specimens from 289 elk collected between December 1991 and January 1993 were examined histologically; no mycobacterial lesions were observed. Lesions of tuberculosis were not detected in tonsils or lymph nodes of the head from an additional 99 hunter-killed, adult elk from one area (area 2) collected in January 1995. Neither M. bovis nor M. paratuberculosis were isolated from any of the specimens cultured. Antibodies to Brucella abortus were detected in serum samples from 0%, 1%, and 1% of elk from three areas sampled (areas 1, 2 and 3), respectively. Brucella abortus biovar 1 was isolated from multiple tissues from one seropositive animal from area 3. Larvae with morphology consistent with Dictyocaulus sp. were found in 12%, 14%, and 0% of fecal specimens tested from areas 1, 2, and 3, respectively. Pasteurella multocida and Actinomyces pyogenes were isolated from a lung with purulent bronchopneumonia and abscesses.