Biomedical subjects
Publications and source records attributed to J L Jarnagin.
Interest in tuberculosis in elephants has been increasing over the past several years in the United States. Several techniques have been used to diagnose mammalian tuberculosis. Currently, the test considered most reliable for diagnosis of TB in elephants is based on the culture of respiratory secretions obtained by trunk washes.
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.
Seventy-nine Mycobacterium bovis isolates recovered from Mexican and Texas cattle were categorized into 16 and 25 distinct types on the basis of IS6110 and direct-repeat fingerprint patterns, respectively. By using a combination of both fingerprint patterns, 30 distinct restriction fragment length polymorphism types were defined. Fifty-eight of 79 isolates (73%) were distributed among nine clusters. Clustered isolates were identified within herds, as well as in geographically disperse herds in Texas and Mexico. This observation is consistent with active transmission within herds and among herds, presumably as a result of active or historical cattle movements. The majority of bovine isolates (64 of 79) exhibited a single copy of IS6110. Interestingly, in contrast to previous studies, a high percentage of bovine isolates (15 of 79) exhibited multiple IS6110 copies (two to five) distributed among 11 different restriction fragment length polymorphism types. It is speculated that transmission from noncattle sources may be responsible. Continued fingerprinting of isolates originating from nonbovine sources and herd surveys is expected to provide useful information regarding the epidemiology of tuberculosis in this region.
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.
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.
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One hundred eighty-four isolates representing 23 species of mycobacteria were identified using computer-assisted analysis. All isolates were examined using a standard series of 12 biochemical tests. These tests were selected because of their reproducibility and ease of performance in the laboratory. Data from these tests were analyzed by a computer that had been previously programmed to process the information and make a species determination. Identifications from the probability model were compared to identifications from conventional methods. There was 96.7% agreement between the 2 methods. The computer-assisted data analysis for identification provides increased accuracy over conventional methods because a statistical probability is applied. It also requires less time. Differences in computer data between mycobacterial species are discussed.
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Lipid analysis was performed on 204 isolates of mycobacteria, using miniaturized thin-layer chromatography (MTLC). A previously described lipid component was found in 92 (96.8%) of 95 isolates of Mycobacterium bovis and in 2 (1.8%) of 109 isolates of other mycobacteria. Using MTLC, an isolate could be identified as M bovis 3 to 4 weeks earlier than by use of conventional biochemical techniques. Examination of isolates for colonial and cellular morphologic characteristics was a useful adjunct in the identification of M bovis by use of MTLC.
Lipid analyses were performed on 107 isolates of mycobacteria, using thin-layer chromatography. A distinctive blue-gray spot was observed after spraying and charring at an Rf of 0.75 for 35 of 38 Mycobacterium bovis isolates examined. The spot, designated M bovis identifying lipid, was observed only with M bovis and was not seen on any other of 9 mycobacterial species examined. This procedure has a high rate of agreement (97.2%) with standard biochemical tests and can rapidly detect M bovis from naturally occurring infections. Although chemical and antigenic characteristics are not fully elucidated, the apparent specificity of this spot for M bovis represents a simple timesaving method for differentiating this species from other mycobacteria when compared with conventional methods presently used.
The isolation of 6 strains of Mycobacterium kansasii from bovine lymph nodes is reported. Occasional isolations of this organism from cattle lymph node lesions provides further evidence of its pathogenicity in domestic animals. The isolates were similar as determined by biochemical and serologic examinations. Histopathologic changes induced by this organism relative to those by M bovis and factors regarding source of infection are discussed.
A microagglutination test was used to identify Brucella dominant antigens from 400 Brucella and non-Brucella cultures. There was 100% agreement between microagglutination and tube agglutination tests in identifying dominant antigen.
Tubercles were seen in the spleen of a male dusky langur (Presbytis obscurus) on laparotomy. Subsequently, tuberculous lesions in the intestine, lungs, and a hilar lymph node were observed on necropsy of the monkey. Histologic examinations of these tissues revealed granulomas, and acid-fast bacilli were observed within granulomas in replicate sections that were stained with auramine-O. An acid-fast organism was isolated and identified as Mycobacterium bovis. Guinea pigs and rabbits inoculated intraperitoneally with the organism developed granulomas in the lungs, liver, and spleen. Lesions did not develop in chickens inoculated with the culture.
Fennec foxes (Fennecus zerda) in 2 zoos were found on necropsy to have lesions typical of those found in canine tuberculosis. Histologic examination revealed numerous acid-fast bacilli in lesions of liver, portal lymph node, spleen, kidney, and lung. Mycobacterium bovis isolated from tissues was identified by biochemical methods and by pathogenicity tests in guinea pigs and rabbits.
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A method for evaluating the viability of mycobacterial cultures using fluorescein diacetate and ethidium bromide is described. Smears from 2-4 week old cultures of mycobacteria were stained with a 0.005% Dubos albumin broth dilution of fluorescein diacetate for thirty minutes at 37 C. The smears were counterstained with a 0.005% aqueous solution of ethidium bromide for ten minutes at room temperature and blotted dry. One drop of glycerol was placed on the smear and covered with a 22 X 40 mm coverslip. Stained smears were observed with fluorescence microscopy at 450 X. Viable organisms hydrolyzed the fluorescein diacetate and appeared yellow-green while dead organisms absorbed the ethidium bromide and appeared red-orange. Aliquots of material from which the slides were made were concurrently placed on Lowenstein-Jensen media and cultured for growth as a confirmation of viability. Investigation of 104 mycobacterial isolates using the described test showed that the viability of mycobacterial cultures can be determined by this method.