Abstract.A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex ( M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.The United States has been actively pursuing the goal of bovine tuberculosis eradication for almost a century, and that objective has nearly been achieved. Most of the initial progress was made by using the caudal fold skin test to identify infected herds, which were then eliminated, but current regulatory efforts are focused on surveillance methods to identify infected animals. The cases are then traced to the herd of origin for further testing or other appropriate resolution of the problem.Tuberculosis surveillance depends on detection of gross lesions by regulatory officials in meat inspection facilities or by pathologists in state diagnostic laboratories. In some cases, suspect tissues are obtained from animals other than domestic cattle, usually farmed elk (Cervus elaphus) and deer, but occasionally from other captive ruminant species or from free-ranging wildlife. Formalin-fixed tissue samples are routinely submitted for histopathologic examination, and in most

Abstract. Sections of tuberculous lesions from 23 elk (Cervus elaphus nelsoni) and red deer (Cervus elaphus elaphus), 12 fallow deer (Dama dama), 10 sika deer (Cervus nippon), and 30 cattle were examined and compared.Lesions were scored for caseous necrosis, mineralization, neutrophils, macrophages, giant cells, and acid-fast bacilli. Some differences in lesion morphology between the species were noted. ElMred deer lesions had marked variation and often differed from bovine lesions in several characteristics; elMred deer lesions usually had scattered peripheral mineralization rather than central mineralization and contained more neutrophils and fewer giant cells than did bovine lesions. Fallow deer lesions contained more giant cells but were otherwise indistinguishable from elk lesions. Sika deer lesions had more giant cells and fewer neutrophils than did lesions from cattle or other cervid species. Sika deer giant cells were larger and contained more nuclei than did giant cells in the other species.

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.

Abstract. A Mycobacterium bovis-infected herd of captive wapiti (Cervus elaphus nelsoni) in Colorado wasdepopulated after lesions of bovine tuberculosis were confirmed in 8 of 10 tuberculin skin test reactors. Of the 43 animals > 1 year of age, 26 had gross lesions suggestive of tuberculosis, 24 had microscopic lesions of tuberculosis, and 23 had acid-fast bacilli associated with the lesions. Lungs and retropharyngeal lymph nodes were the most frequently affected sites. Most lesions grossly and microscopically resembled tuberculosis in cattle; however, some lesions resembled abscesses or ovine caseous lymphadenitis lesions. Special stains and immunohistochemical techniques labeled few to numerous mycobacteria associated with the lesions.Until recently, bovine tuberculosis was considered an unusual and sporadic disease in cervids. Reported cases involved captive and wild cervids and were often attributed to contact with infected cattle or bison. 14,16,24 With recent development and expansion of game farming, the prevalence, economic impact, and public health significance of tuberculosis in captive cervids have greatly increased. 6,9,32 In New Zealand, tuberculosis was diagnosed in wild red deer (Cervus elaphus elaphus) in 1970 2 and in farmed deer in 1978. 17 It is now considered the most important bacterial disease in New Zealand's farmed deer population. 6,13,32 Bovine tuberculosis has also been diagnosed in several species of free-living and captive cervids from other countries 1,8,11,15,19,20,25,[29][30][31]33,35,36,43 In North America, reports of tuberculosis in cervids have been limited to sporadic cases in wild and captive white-tailed deer (Odocoileus virginianus) 26 This report provides more detailed descriptions of all gross and microscopic lesions of bovine tuberculosis observed in this herd. Materials and methodsAll animals were killed by gunshot. Necropsies were done, gross lesions recorded and photographed, and tissue specimens collected.26 Samples of grossly observed lesions and/ or parotid, mandibular, retropharyngeal, mediastinal, bronchial, hepatic, and mesenteric lymph nodes that were routinely bisected were placed in 10% neutral buffered formalin and saturated sodium borate transport solution for mycobacterial culture. Specimens were transported within 4 days to the National Veterinary Services Laboratories, Ames, Iowa, for histopathologic examination and mycobacterial culture.Formalin-fixed tissues were routinely processed using paraffin embedding, and 5-µm sections were stained with hematoxylin and eosin (HE). Significantly mineralized tissues were decalcified and routinely processed. Additional sections from tissues with lesions suggestive of tuberculosis were stained by a new fuchsin-methylene blue procedure (NF) 18 and acridine orange-auramine O (AOA0) 27 and were examined for the presence of acid-fast bacilli and AOAO fluorescent bacilli. Some sections were also stained with Giemsa and Gram's stains.Selected specimens were stained by a labeled streptavidinbiotin immunohistochemical t...

Abstract. Specimens of blood, lymph nodes, spleens, and genitalia were collected at slaughter from seven 3-and 4-year-old male bison that had recently become seropositive for brucellosis. The animals were from a captive herd of approximately 3,500 bison located in central South Dakota. Brucella abortus biovar 1 was isolated from 2 or more specimens from each of 6 bison. Severe necrotizing and pyogranulomatous orchitis was present in 1 testicle from 1 bull, and 4 animals had mild to marked seminal vesiculitis. Immunohistochemical staining labeled organisms in seminal vesicles and the testicle with orchitis. Ultrastructurally, intact bacilli were present in cytoplasmic vacuoles of some macrophages; other macrophages contained intracytoplasmic aggregates of calcified coccobacilli.Brucella abortus produces abortion in cattle, bison, 9,19,23 and elk. 21 Additionally, metritis and retained placentas are associated with the infection in cattle and bison. 7,23 In male cattle, brucellosis typically is associated with seminal vesiculitis, orchitis, and epididymitis. There are few reports of similar lesions in the genitalia of B. abortus-infected bison bulls. 7,8,22,23 The purpose of this report is to describe the sites of bacterial localization and lesions associated with natural infection by B. abortus in 3-and 4-year-old bison bulls from a large captive herd in South Dakota. Materials and methodsBlood and tissue specimens including lymph nodes, spleen, and genitalia were collected from seven 3-and 4-yrold bison bulls at slaughter (Table 1). The bulls were from a herd of 3,500 bison located in central South Dakota. The herd was under state quarantine and had been engaged in a whole-herd test and slaughter program since 1990. In December 1994, 80, 3-and 4-yr-old bulls and 4 adult cows were seropositive on standard serologic tests for brucellosis. These animals had been seronegative on all previous tests; the last test had been in the spring of 1994. All of the seropositive animals were slaughtered for meat.Portions of testicles, epididymides, and seminal vesicles were removed and placed in 10% neutral buffered formalin.