Induction of PrPSc-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease.

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrPC into PrPSc. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrPSc-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrPSc-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.

Protective effect of EC-18, a synthetic monoacetyldiglyceride on lung inflammation in a murine model induced by cigarette smoke and lipopolysaccharide.

The antler of Sika deer (Cervus nippon Temminck) has been used a natural medicine in Korea, China and Japan, and a monoacetyldiaglyceride (1-palmitoyl-2-linoleoyl-3-acetylglycerol, PLAG) was found in the antler of Sika deer as a constituent for immunomodulation. In this study, we investigated protective effects of EC-18 (a synthetic copy of PLAG) on inflammatory responses using a cigarette smoke with lipopolysaccharide (LPS)-induced airway inflammation model. Mice were exposed to cigarette smoke for 1h per day for 3days. Ten micrograms of LPS dissolved in 50µL of PBS was administered intra nasally 1h after the final cigarette smoke exposure. EC-18 was administered by oral gavage at doses of 30 and 60mg/kg for 3days. EC-18 significantly reduced the number of neutrophils, reactive oxygen species production, cytokines and elastase activity in bronchoalveolar lavage fluid (BALF) compared with the cigarette smoke and LPS induced mice. Histologically, EC-18 attenuated airway inflammation with a reduction in myeloperoxidase expression in lung tissue. Additionally, EC-18 inhibited the phosphorylation of NF-κB and IκB induced by cigarette smoke and LPS exposure. Our results show that EC-18 effectively suppresses neutrophilic inflammation induced by cigarette smoke and LPS exposure. In conclusion, this study suggests that EC-18 has therapeutic potential for the treatment of chronic obstructive pulmonary disease.

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil / Ocorrência de anticorpos anti -Toxoplasma gondii, Neospora caninum e Leptospira interrogans em um rebanho de cervídeos de cativeiro do sul do Brasil

Abstract A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

Resumo Um grande número de zoológicos brasileiros abriga espécies de cervídeos ameaçados de extinção, entretanto, estudos de vigilância de doenças em cervídeos de cativeiro são escassos. Amostras de sangue de 32 cervídeos brasileiros (Blastocerus dichotomus, Mazama nana e Mazama americana), mantidos em cativeiro no Refúgio Biológico Bela Vista (Foz do Iguaçu, Brasil), foram investigados para 10 patógenos de ruminantes, visando monitorar o estado de saúde dos cervídeos e avaliar a presença de agentes zoonóticos. As amostras de soro foram testadas para Brucella abortus, Leptospira (23 sorovares), Toxoplasma gondii, Neospora caninum, diarreia viral bovina, rinotraqueíte infecciosa bovina, febre aftosa, encefalomielite equina do oeste, encefalomielite equina do leste e encefalomielite equina venezuelana. Foram detectados anticorpos para T. gondii (15,6%), N. caninum (6,2%) e para L. interrogans sorogrupo Serjoe (3,1%). As sorologias apresentaram resultado negativo para as demais doenças. Os cervídeos foram considerados clinicamente sadios e assintomáticos para doenças. Comparados aos estudos de populações de vida livre, as soroprevalências para os mesmos agentes testados foram menores para os cervídeos mantidos no Refúgio, indicando as boas condições sanitárias e a qualidade das práticas de manejo no zoológico.

Oral vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis Bacillus Calmette-Guerin (BCG).

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer, thus interfering with the intraspecies and interspecies transmission cycles. Thirty-three white-tailed deer were assigned to one of two groups; oral vaccination with 1 × 10(8) colony-forming units of M. bovis BCG Danish (n = 17); and non-vaccinated (n = 16). One hundred eleven days after vaccination deer were infected intratonsilarly with 300 colony-forming units of virulent M. bovis. At examination, 150 days after challenge, BCG vaccinated deer had fewer gross and microscopic lesions, fewer tissues from which M. bovis could be isolated, and fewer late stage granulomas with extensive liquefactive necrosis. Fewer lesions, especially those of a highly necrotic nature should decrease the potential for dissemination of M. bovis within the host and transmission to other susceptible hosts.

Global gene expression profiling of monocyte-derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection.

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johne's disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype. Cells were infected with MAP in vitro and gene expression analysed by RNA-Seq. Transcriptome analysis revealed a more disrupted gene expression profile in macrophages from susceptible animals compared with cells from resistant animals in terms of the number of genes up- or downregulated. Highly upregulated genes were related to chemotaxis (CXCL10, CSF3, and CCL8) and type 1 interferon signalling (RSAD2, IFIT1, IFIT2, ISG12, ISG15, USP18, and HERC6). Upregulation of these genes was observed to be greater in macrophages from susceptible animals compared to cells from resistant animals in response to in vitro MAP infection. These data support the use of transcriptomic approaches to enable the identification of markers associated particularly with susceptibility to MAP infection.

Production of a bovine IL-12p40 probe and application using in situ hybridization on ruminant fixed tissues.

Pro-inflammatory cytokines (particularly IL-12) are important for initiating protective T helper 1 (Th1)-type immune responses and hence vital for combating intracellular infections and tumours. In situ hybridization (ISH) provides a powerful diagnostic tool allowing the identification and localization of cells producing these mediators in fixed tissues. The objective of this work was to produce a bovine IL-12p40 probe that allows detection of IL-12p40 mRNA in fixed tissues from different ruminant species. The RNA probe sequence is 447bp in length and from a region with high cross-species-sequence homology (>97.3% homology) to the ovine, cervine, caprine and bubaline IL-12p40 genes. ISH was carried out on paraformaldehyde fixed tissues collected from cattle, sheep and goats. The probe was efficient in identifying IL-12p40 expressing cells in fixed tissues from all these species. In conclusion, the IL-12p40 probe was efficient in identifying and localizing cells that express IL-12p40, and provides a good immuno-diagnostic technique to characterize immune responses in fixed tissues.

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected fallow deer (Dama dama).

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis.

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.

Contraceptive vaccines for wildlife: a review.

Wildlife, free-ranging and captive, poses and causes serious population problems not unlike those encountered with human overpopulation. Traditional lethal control programs, however, are not always legal, wise, safe, or publicly acceptable; thus, alternative approaches are necessary. Immunocontraception of free-ranging wildlife has reached the management level, with success across a large variety of species. Thus far, the immunocontraceptive research and management applications emphasis have been centered on porcine zona pellucida and gonadotropin-releasing hormone vaccines. Contraceptive success has been achieved in more than 85 different wildlife species, at the level of both the individual animal and the population. At the population management level with free-ranging species, the primary focus has been on wild horses, urban deer, bison, and African elephants. The challenges in the development and application of vaccine-based wildlife contraceptives are diverse and include differences in efficacy across species, safety of vaccines during pregnancy, the development of novel delivery systems for wild and wary free-ranging animals, and the constraints of certain non-contraceptive effects, such as effects on behavior. Beyond the constraints imposed by the public and a host of regulatory concerns, there exists a real limitation for funding of well-designed programs that apply this type of fertility control.

A new Beta defensin from sika deer: molecular cloning and sequence characterization.

The beta-defensins are small, well-characterized peptides with broad antimicrobial activities. Here we report the identification of a novel ß-defensin, sika deer ß-defensin-1 (siBD-1), from sika deer tissues with a pair of PCR primers according to the conserved cDNA sequences of known ruminant ß-defensins. Total RNA was extracted from the tongue epithelia of a sika deer and the 418 bp cDNA encoding siBD-1 was amplified by the reverse transcription PCR (RT-PCR), 5'- and 3'-RACE. The cDNA contained an open reading frame (ORF) of 192 bases which encoded a 64 amino acid prepro-peptide. The prepro-peptide contained six invariantly spaced cysteine residues, which is the ß defensin consensus sequence. The putative mature peptide of the siBD-1 contained nine positively charged residues (5 arginine-R, 3 lysine-K, and 1 histidine-H). The sequence homology shows that siBD-1 has 73.0-90.6% amino-acid identity and 74.6-90.6% cDNA identity with other ruminant beta-defensins, sharing the greatest identity with buffalo enteric ß-defensin in both amino acid and nucleotide sequences.

Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer.

Although the causative agent of Johne's disease, Mycobacterium avium subsp. paratuberculosis, is well known, the etiology of disease and the immune responses generated in response to infection are still poorly understood. Knowledge of definitive markers of protective immunity, infection, and the establishment of chronic granulomatous Johne's disease is necessary to advance vaccine and diagnostic development. We sought to profile the immune responses occurring within jejunal lymph nodes of experimentally challenged red deer (Cervus elaphus). Quantitative PCR was utilized to measure a range of cytokines, signaling molecules, and transcription factors involved in Th1, Th2, Treg, and Th17 immune responses. Significant differences in gene expression were observed between control, minimally diseased, and severely diseased animals, with severely diseased animals showing elevated proinflammatory transcripts and reduced anti-inflammatory transcripts. We identified a proinflammatory cytokine milieu of gamma interferon, interleukin-1α (IL-1α), and IL-17, which may contribute to the immunopathology observed during clinical Johne's disease and suggest that Th2 and Treg immune responses may play an important role in controlling the development of immunopathology in infected animals.

Molecular cloning of CD18 of bison, deer and elk, and comparison with that of other ruminants and non-ruminants.

Pneumonia caused by Mannheimia haemolytica is an important disease of cattle, domestic sheep, bighorn sheep and goats. Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these species. Lkt utilizes CD18, the beta subunit of beta(2)-integrins, as its functional receptor on leukocytes of these species. Cytotoxicity assays revealed that leukocytes from bison, deer, and elk are also susceptible to Lkt-induced cytolysis. The availability of cDNA encoding CD18 of bison, deer and elk would facilitate the comparison of a greater number of ruminant CD18 cDNA with that of non-ruminants as a means of the elucidation of the molecular basis for the specificity of M. haemolytica Lkt for ruminant leukocytes. Herein, we report the cloning and characterization of bison, deer, and elk CD18. The full-length cDNA of bison and deer consists of 2310bp with an ORF encoding 769 amino acids while elk CD18 consists of 2313bp with an ORF encoding 770 amino acids. This gene is highly conserved among ruminants compared with non-ruminants. Phylogenetic analysis based on amino acid sequences showed that CD18 of bison is most closely related to that of cattle while CD18 of deer and elk are more closely related to each other.

The single-shot GnRH immunocontraceptive vaccine (GonaCon) 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. METHOD OF STUDY: Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments. RESULTS: The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine. CONCLUSION: One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.

Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacillus Calmette Guerín.

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt the cycle of deer to deer and deer to cattle transmission. Thirty-one white-tailed deer were assigned to one of three groups; 2 SC doses of 10(7)CFU of M. bovis BCG (n=11); 1 SC dose of 10(7)CFU of M. bovis BCG (n=10); or unvaccinated deer (n=10). After vaccination, deer were inoculated intratonsilarly with 300 CFU of virulent M. bovis. Gross lesion severity scores of the medial retropharyngeal lymph node were significantly reduced in deer receiving 2 doses of BCG compared to unvaccinated deer. Vaccinated deer had fewer lymph node granulomas than unvaccinated deer, and most notably, fewer late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli. BCG was isolated from 7/21 vaccinated deer as long as 249 days after vaccination. In one case BCG was transmitted from a vaccinated deer to an unvaccinated deer. In white-tailed deer BCG provides measurable protection against challenge with virulent M. bovis. However, persistence of vaccine within tissues as well as shedding of BCG from vaccinates remain areas for further investigation.

Experimental persistent infection with bovine viral diarrhea virus in white-tailed deer.

Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.

Cervine (Cervus elaphus) cytokine mRNA quantification by real-time polymerase chain reaction.

It has been difficult to perform cytokine studies for many wildlife and nontraditional species because of a lack of immunologic reagents at the protein level. Recently, simple and rapid assays for quantifying mRNA expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) have been used for analysis of cytokine profiles in humans and other mammalian species. This report describes the development and application of real time RT-PCR to measure the expression of several important elk (Cervus elaphus) cytokine mRNAs, including interleukin (IL)-2, IL-4, IL-10, IL-12p40, interferon-gamma, tumor necrosis factor (TNF)-alpha, and the enzyme-inducible nitric oxide synthase, all of which are involved in immune responses and regulation. For the broadest potential application of the assay, primers and probes were designed using consensus sequences from several species of interest. To obtain standardized quantitative results, external controls consisting of a DNA template for each target gene were used to generate linear standard curves over a 6 to 8 log range with detection of as few as 10 copies of amplicon per reaction. Sample-to-sample variation in the efficiency of the RT, as well as in the quantity and quality of the starting RNA, was compensated for by normalizing the results to the endogenous housekeeping gene beta(2)-microglobulin. The assay was evaluated by monitoring the kinetics of cytokine mRNA synthesis induced by mitogenic and antigenic stimulation of peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis-infected elk. Concanavalin A-stimulated PBMCs demonstrated a rapid but transient increase in cytokine mRNA expression following in vitro mitogenic activation with optimal mRNA induction observed after 4 to 16 hr. The PBMCs stimulated with the mycobacterial recall antigen, bovine-purified protein derivative (PPD-bovis), demonstrated variable mRNA induction kinetics for each cytokine. Whereas PPD-bovis optimally induced IL-2 mRNA after 8 hr of in vitro stimulation, longer in vitro stimulation times were necessary for the optimal induction of IL-4 and TNF-alpha mRNA (up to 48 hr). We demonstrate real-time RT-PCR to be a rapid, sensitive, and reproducible technique, which will make it a valuable tool in the study of immunologic responses and cytokine profiles of cervids and other nontraditional livestock and wildlife species.

In search of the active PZP epitope in white-tailed deer immunocontraception.

Native porcine zona pellucida (PZP) has been shown to be highly effective as an immunocontraceptive in white-tailed deer. However, the immunogenicity of PZP extracted from pig ovaries may vary from lot to lot and the extract has the potential of containing either viral or pathogenic material. Determination of the immunocontraceptive epitopes of PZP would allow portions of the molecule to be synthesized or inserted into a recombinant system for production of a consistent and safe vaccine. In this study, epitopes of PZP were selected and tested by in vitro binding, immunogenicity in rabbits, immunogenicity and immunocontraception in deer. Sera from PZP immunocontracepted deer were tested on ELISA plates containing immobilized peptides from ZP1 and ZP3alpha. Peptides with which sera from infertile deer reacted (six peptides from ZP1 and six peptides from ZP3alpha) were selected, synthesized and tested for immunogenicity in rabbits. Deer were then immunized with combinations of peptides from either the ZP1 or ZP3alpha groups. ZP3alpha peptides induced high immune titers against native PZP, but did not induce infertility in the deer. Although ZP1 peptides induced lower titers, deer immunized with two ZP1 peptides exhibited multiple estrus events and infertility, typical of that for deer immunized with native PZP vaccine. Competitive inhibition assays using the ZP1 peptides demonstrated that the peptide comprising pins 10-16 was most effective in blocking binding by the serum antibody of native PZP immunized deer. This peptide was used to immunocontracept deer, resulting in a significant reduction in fawning for 1 year.

Cell mediated and humoral immune responses of white-tailed deer experimentally infected with Mycobacterium bovis.

The objective of this study was to improve the understanding of immune responses of whitetailed deer (Odocoileus virginianus) infected with Mycobacterium bovis. Ten mature, female, white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3)or 2 x 10(5)colony-forming units of M. bovis. Lymphocyte proliferation and humoral response to M. bovis PPD and the M. bovis protein, MPB70 were measured. Deer were tested for exposure to M. bovis by the comparative cervical skin test. Biopsy specimens of skin test sites were examined microscopically and immunohistochemically. The comparative cervical skin test correctly identified all M. bovis -inoculated deer as exposed to M. bovis. Lymphocyte proliferative responses to MPB70 were more consistent than responses to M. bovisPPD in M. bovis -inoculated deer. Antibody responses were more prominent in deer with disseminated disease than in deer with localised disease. The cellular components of delayed-type hypersensitivity reactions at skin test sites were similar to tuberculin reactions in other species. T lymphocytes of the gamma/delta phenotype were seen in increased numbers in M. bovisPPD injection sites.

Evaluation of low-level aflatoxin in the diet of white-tailed deer.

We evaluated the response of white-tailed deer (WTD) (Odocoileus virginianus) to dietary aflatoxin. Fourteen 4-to-5-mo-old WTD were used in this 8-wk study, conducted between November 1993 and January 1994. Seven animals received a ration containing 800 parts per billion (ppb) total aflatoxin (AF). Seven control animals received the same ration without AF. At 0, 1, 3, 6 and 8 wk, feed consumption, feed conversion, liver enzymes, bile acid levels, and immune function via lymphocyte proliferation assays and delayed type hypersensitivity reactions were determined. At the conclusion of the 8-wk feeding trial, deer were euthanized and necropsied. Clinical illness was not evident in any of the animals, but by the end of the study, AF-fed deer had reduced feed consumption and body weight as compared to control deer; the differences were not statistically significant. The AF-exposed group had a significant increase (P = 0.03) in serum bile acid concentration as compared to control deer. Two AF-exposed deer had gross and histologic hepatic lesions indicative of a mild degenerative hepatopathy. Residues of an aflatoxin metabolite, aflatoxin M1, were found in the livers of all treated animals. No differences in immune function were detected between the two groups. We conclude that consumption of 800 ppb AF in the diet of young WTD over an 8-wk period can produce subclinical hepatic injury.

Macrophage function in deer.

Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis. Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells. Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested. The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level. Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h. Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation. Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG). Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens. These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.