Jack C. Rhyan

Detection of Mycobacterium Bovis in Formalin-Fixed, Paraffin-Embedded Tissues of Cattle and Elk by PCR Amplification of an IS6110 Sequence Specific for Mycobacterium Tuberculosis Complex Organisms:

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.

Evidence of Brucella infection in Parafilaroides lungworms in a Pacific harbor seal (Phoca vitulina richardsi).

Multiple isolates of Brucella sp. that differ from the recognized species within this genus have recently been isolated from viscera of 4 harbor seals (Phoca vitulina), 2 harbor porpoises (Phocoena phocoena), 1 common dolphin (Delphinus delphis), an Atlantic white-sided dolphin (Lagenorhynchus acutus), 2 striped dolphins (Stenella coeruleoalba), a hooded seal (Cystophora cristata), and a gray seal (Halichoerus grypus), 9,18 all from the Scottish coast. A similar Brucella sp. was isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) from the coast of Califomia. During routine capture operations, 18 of 102 Pacific harbor seals (Phoca vitulina richardsi) and 4 of 50 California sea lions (Zalophus californianus) from Puget Sound, Washington, had been reported with positive titers to Brucella sp. In this report, we describe the isolation, tissue location, and immunohistochemical and ultrastructural features of Brucella sp. infection in a pacific harbor seal. These findings suggest that transmission of brucellosis by infected lungworms is a possibility. On March 28, 1996, a 7-9-month-old 17.5-kg male Pacific harbor seal (105 cm length, snout to tail tip) was collected dead from Restoration Point on Bainbridge Island in the Puget Sound, Washington. The seal had been dead for 2-5 days, and there was slight decomposition. The carcass was transported to the Washington Department of Fish and Wildlife Marine Mammal Investigations Laboratory in Tacoma and necropsied. Blood drawn from the heart was separated by centrifugation, and serum was sent to the Washington Department of Agriculture Laboratory to screen for antibodies to Brucella sp. Values were compared with the standards outlined in the Brucellosis Eradication Uniform Methods and Rules (1992, revised 1994, USDA, APHIS). The sample was tested using B. abortus antigens provided by the National Veterinary Services Laboratory (NVSL), Ames, Iowa. Official protocols for

Immunocontraception of florida feral swine with a single-dose GnRH vaccine

Methods to limit fertility of feral swine are needed to reduce transmission of diseases and agricultural and ecosystem damage.

The Single-Shot GnRH Immunocontraceptive Vaccine (GonaCon TM ) in White-Tailed Deer: Comparison of Several GnRH Preparations

Problem  An effective, single‐injection, multi‐year, GnRH contraceptive agent is needed to control reproduction in overabundant white‐tailed deer populations.

Contraception of bison by GnRH vaccine: a possible means of decreasing transmission of brucellosis in bison.

Preventing pregnancy in brucellosis-infected bison (Bison bison) provides a potential means of preventing transmission of disease. To determine whether a gonadotropin-releasing hormone (GnRH) vaccine was effective in reducing pregnancy in bison and to study the safety of injecting GnRH in pregnant bison, a study was conducted at the Idaho Fish and Game Wildlife Health Laboratory in Caldwell, Idaho (USA). Four pregnant and two nonpregnant female bison were given a single injection of GnRH vaccine, and five pregnant adult females were given a sham injection that contained only adjuvant. Three of the GnRH-vaccinated bison that were pregnant at the time of vaccination delivered healthy calves. One treated bison had dystocia that resulted in a dead calf. All control bison delivered healthy calves. After calving, females of both groups were exposed to two bulls. Treated bison were palpated 6 wk after exposure to the bulls, and blood was drawn for pregnancy-specific protein B analysis. The six treated bison were not pregnant. The sham-treated bison became pregnant and delivered viable calves. This study demonstrates that a single dose of GnRH vaccine is effective in preventing pregnancy in female bison for at least 1 yr.

Brucella Suis Biovar 1 in Naturally Infected Cattle: A Bacteriological, Serological, and Histological Study

and immunodiffusion for serodiagnosis of paratuberculosis. Can J Vet Res 53:405–410. 20. Wright PF, Kelly WA, Gall DEJ: 1985, Application of a timing protocol to the reduction of interplate variability in the indirect enzyme immunoassay for detection of anti-Brucella antibody. J Immunoassay 6:189–205. 21. Yokomizo Y, Merkal RS, Lyle PAS: 1983, Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis. Am J Vet Res 44:2205–2207. 22. Yokomizo Y, Yugi H, Merkal RS: 1985, A method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine paratuberculosis Jpn J Vet Sci 47:111–119.

PATHOLOGY OF BRUCELLOSIS IN BISON FROM YELLOWSTONE NATIONAL PARK

Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus-infected herd in July and August, 1997. Results of the bison and cattle studies were similar.

PATHOGENESIS AND EPIDEMIOLOGY OF BRUCELLOSIS IN YELLOWSTONE BISON: SEROLOGIC AND CULTURE RESULTS FROM ADULT FEMALES AND THEIR PROGENY

Our objective in this prospective study was to determine the natural course of Brucella abortus infection in cohorts of seropositive and seronegative, female bison (Bison bison) and their offspring in Yellowstone National Park (YNP) for 5 yr. We collected specimens from 53 adult females and 25 calves at least once and from 45 adults and 22 calves more than once. Annual seroconversion rates (negative to positive) were relatively high (23% for calves and juvenile bison, 6% in the total sample of adult female bison in our study, and 11% in the adult females that began the study as seronegatives). Antibody was not protective against infection, even for calves that passively received antibody from an infected mother’s colostrum. Antibody levels stayed remarkably constant, with only a slow decline over time. We found only two seroconversions from a weak positive status to negative. Infected bison aborted and shed viable bacteria. Risk of shedding infective Brucella was highest for bison in the 2 yr following seroconversion from negative to positive. In one bison, we detected shedding for 3 yr following seroconversion. Regardless of serostatus of dams and neonates, most calves were seronegative by 5 mo of age. There was no relationship between the antibody status of the dam and the tendency of a calf to seroconvert to positive during the duration of the study.

Reproduction and Survival of Yellowstone Bison

Abstract The conservation of bison (Bison bison) from near extinction to >4,000 animals in Yellowstone National Park has led to conflict regarding overabundance and potential transmission of brucellosis (Brucella abortus) to cattle. We estimated survival and birth rates from 53 radiocollared adult female bison during 1995–2001, and we used calf:adult (C:A) ratios to estimate reproduction with the combined effects of pregnancy, fetal loss, and neonatal mortality during 1970–1997. Annual survival of adult females was high (0.92; 95% CI = 0.87–0.95) and constant. Birth rates differed by brucellosis status and age. Birth rates were 0.40 calves per female (95% CI = 0.15–0.65) for brucellosis-positive 3 year olds, 0.63 (95% CI = 0.39–0.87) for individuals testing negative, and 0.10 (95% CI = 0.00–0.24) for individuals contracting brucellosis that birth year (sero-converters). Birth rates were 0.64 (95% CI = 0.52–0.76) for brucellosis-positive individuals ≥4 years old, 0.81 (95% CI = 0.73–0.89) for brucellosis-negative individuals, and 0.22 (95% CI = 0.00–0.46) for sero-converters. Spring C:A ratios were negatively correlated with snow pack (β = −0.01 to −0.03, R2 = 0.26–0.60, P < 0.05). Growth rate was highly elastic to adult survival (0.51), and juvenile survival (0.36) was 3 times more elastic than fecundity (0.12). Simulations suggested brucellosis eradication via vaccination would result in increased birth rates and a 29% increase in population growth (λ = 1.09), possibly leading to more bison movements outside the park. Our results will help park managers evaluate bison population dynamics and explore consequences of management actions and disease control programs.

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.