Donald S. Davis

EPIDEMIOLOGY AND DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS IN CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract The deaths of two Asian elephants (Elephas maximus) in August 1996 led the United States Department of Agriculture to require the testing and treatment of elephants for tuberculosis. From August 1996 to September 1999, Mycobacterium tuberculosis infection was confirmed by culture in 12 of 118 elephants in six herds. Eight diagnoses were made antemortem on the basis of isolation of M. tuberculosis by culture of trunk wash samples; the remainder (including the initial two) were diagnosed postmortem. We present the case histories, epidemiologic characteristics, diagnostic test results, and therapeutic plans from these six herds. The intradermal tuberculin test, enzyme-linked immunosorbent assay serology, the blood tuberculosis test, and nucleic acid amplification and culture are compared as methods to diagnose M. tuberculosis infection in elephants.

Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle.

Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 × 107 colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 × 107 CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of brucellosis in bison did not significantly differ grossly or histologically from those in cattle. There were six abortions and two nonviable calves in the bison group, as compared to nine abortions in the 12 similarly inoculated cattle. As determined by bacterial isolations, transmission of B. abortus from bison to cattle (five of 12 susceptible cattle became infected) did not differ statistically from cattle to cattle transmission (six of 12 susceptible cattle became infected) under identical conditions. No single serologic test was consistently reliable in diagnosing B. abortus infected bison for 8 wk PE. Multiple testing periods in which the Card test was used in combination with the bison conjugated enzyme linked immunosorbent assay and the hemolysis-in-gel proved to be a useful battery of serologic techniques to diagnose brucellosis in bison after the initial 8 wk PE.

Brucella vaccines in wildlife.

Brucellosis has been known to exist in populations of wildlife since the early part of the 20th century. At the beginning of this century in the US, Brucella abortus is a problem in elk and bison in the Greater Yellowstone Area, B. suis is prevalent in millions of feral swine in most of the southern states, and caribou/reindeer in Alaska are infected with B. suis biovar 4. Brucellosis has been virtually eliminated in domestic livestock in the US after decades of expensive governmental disease prevention, control and eradication programs. Now the most likely source of transmission of brucellosis to humans, and the risk of reintroduction of brucellosis into livestock is from infected populations of free-ranging wildlife. Brucellosis was eradicated from livestock through a combination of testing, vaccination, and removal of infected animals. The use of vaccines to control brucellosis in populations of wildlife and therefore reducing the risk of transmission to humans and livestock has been proposed in several instances. This manuscript reviews research on the use of Brucella vaccines in species of wildlife with emphasis on safety and efficacy.

BRUCELLA ABORTUS IN BISON. II. EVALUATION OF STRAIN 19 VACCINATION OF PREGNANT COWS

Protection against Brucella abortus induced abortion and infection provided by strain 19 (S19) vaccination was evaluated in American bison (Bison bison) Forty-eight pregnant bison were manually inoculated (MI) with S19 vaccine, 44 were ballistically inoculated (BI) with an absorbable hollow pellet containing lyophilized SI9, and 46 were manually injected with buffered saline as non-vaccinated controls (NVC). All bison were Brucella spp. seronegative prior to the experiment, in the second trimester of pregnancy, and were randomly assigned to experimental groups. Approximately 60 days post-vaccination, abortions were observed in the vaccinated bison. Brucella abortus strain 19 was recovered from a bison that had recently aborted, her fetus, and from 11 of 12 other aborted fetuses. Fifty-eight percent (53 of 92) of vaccinated bison aborted, and no abortions were observed in the NVC bison. One cow aborted during her second postvaccinal pregnancy and S19 was identified from the dam and fetus indicating that chronic S19 infections can occur in bison. Positive antibody titers were present 10 mo post-vaccination in 73% (66 of 91) of the bison. Thirteen mo post-vaccination, 30 MI vaccinates, 27 BI vaccinates, and 30 NVC bison were challenged during the second trimester of pregnancy with 1 × 107 CFU of B. abortus strain 2308 via bilateral conjunctival inoculation. Protection against abortion was 67% (P ≤ 0.0001) for vaccinated bison compared to 4% in NVC. Protection against B. abortus infection was determined to be 39% (P ≥ 0.001) for vaccinates and 0% (zero of 30) for NVC. Persistent antibody titers, vaccine induced abortions, and chronic S19 infections indicate that the S19 vaccine doses used in this study are not suitable for pregnant bison.

Characterization of the caprine model for ruminant brucellosis

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Naturally occurring hepatozoonosis in a coyote.

Schizonts of Hepatozoon sp. were found in the myocardium of an adult coyote (Canis latrans) collected from the Aransas National Wildlife Refuge, Austwell, Texas. This constitutes the first time hepatozoonosis has been recorded in Canidae in the Western Hemisphere.

EFFICACY OF SINGLE CALFHOOD VACCINATION OF ELK WITH BRUCELLA ABORTUS STRAIN 19

Abstract Brucellosis has been eradicated from cattle in the states of Wyoming, Montana, and Idaho, USA. However, free-ranging elk (Cervus elaphus) that use feedgrounds in the Greater Yellowstone Area (GYA) and bison (Bison bison) in Yellowstone and Grand Teton national parks still have high seroprevalence to the disease and have caused loss of brucellosis-free status in Wyoming. Management tools to control or eliminate the disease are limited; however, wildlife vaccination is among the methods currently used by wildlife managers in Wyoming. We conducted a controlled challenge study of single calfhood vaccination. Elk calves, caught in January and February of 1999 and 2000 and acclimated to captivity for 3 weeks, were randomly assigned to control or vaccinate groups. The vaccinate groups received Brucella abortus vaccine strain 19 (S19) by hand-delivered intramuscular injection. Calves were raised to adulthood and bred at either 2.5 or 3.5 years of age for 2000 and 1999 captures, respectively. Eighty-nine (44 controls, 45 vaccinates) pregnant elk entered the challenge portion of the study. We challenged elk at mid-gestation with pathogenic B. abortus strain 2308 by intraconjunctival instillation. Abortion occurred in significantly more (P = 0.002) controls (42; 93%) than vaccinates (32; 71%), and vaccine protected 25% of the vaccinate group. We used Brucella culture of fetus/calf tissues to determine the efficacy of vaccination for preventing infection, and we found that the number of infected fetuses/calves did not differ between controls and vaccinates (P = 0.14). Based on these data, single calfhood vaccination with S19 has low efficacy, will likely have only little to moderate effect on Brucella prevalence in elk, and is unlikely to eradicate the disease in wildlife of the GYA.

CONSERVATION OF GERM PLASM FROM BISON INFECTED WITH BRUCELLA ABORTUS

Reproductive procedures for cattle were adapted to American bison (Bison bison) to evaluate the potential preservation of germ plasm from bison infected with Brucella abortus without transmission of the pathogen to the recipient or offspring. Two of four experimentally inoculated bison bulls excreted B. abortus in the semen. Four healthy calves were produced from non-infected, un-vaccinated bison cows by natural breeding with a bison bull excreting B. abortus in the semen. There was no seroconversion of the cows or their calves. Two culture negative bison calves were produced by superovulation of infected bison donor cows followed by artificial insemination and embryo transfer without transmitting B. abortus to recipient cows or calves. These limited data indicate that embryo manipulatory procedures and natural breeding in bison may facilitate preservation of valuable germ plasm from infected bison while reducing the risk of transmission of B. abortus to recipients and progeny.

Genome-Wide Polymorphism and Comparative Analyses in the White-Tailed Deer (Odocoileus virginianus): A Model for Conservation Genomics

The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms.

Safety of Brucella abortus Strain RB51 in Bison

To determine the safety of Brucella abortus strain RB51 (SRB51) vaccine in American bison (Bison bison), 31 animals from a herd with brucellosis were used. In October 1996, 10 adult bison males and seven calves were vaccinated with the standard calfhood cattle dose of 1.8 × 1010 colony forming units (CFU) of SRB51 subcutaneously while the adult females received the standard adult cattle dose of 1 × 109 CFU. Western immunoblot indicated the presence of SRB51 antibodies following vaccination. To evaluate prolonged bacterial colonization of tissues, the adult males, calves, and three adult females were divided into two groups which were slaughtered at either 13 or 16 wk post-vaccination. At necropsy, tissue samples were obtained for B. abortus culture from the liver, spleen, lymph nodes, and reproductive tract of each animal. While B. abortus field strain was cultured from one adult bull, no SRB51 was isolated from any of the animals. Seven pregnant females were monitored until parturition for signs of abortions and fetal lesions. Six cows delivered healthy calves and one delivered a dead full-term calf that was brucellae negative. Based on these results, administration of SRB51 to bison did not cause prolonged bacterial colonization of tissues in calves, adult males, or adult females. Furthermore, SRB51 did not induce abortions following vaccination in the second month of gestation.