Comparative effects of GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines for controlling reproduction in white-tailed deer (Odocoileus virginianus).

Fawning rates and mating behaviour were compared between white-tailed deer (Odocoileus virginianus) treated with GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines from 1997 to 2000. Female deer from a herd of 102 deer at Seneca Army Depot, near Romulus, New York, were treated with prime and booster injections of PZP (n = 22) or GnRH vaccine (n = 32), or remained untreated as controls (n = 34). During the summers after booster treatment, observed fawning rates for adult female deer were similar for both PZP-treated (0.10-0.11 fawns per female) and GnRH-treated (0.13-0.22 fawns per female) female deer, and were significantly lower (t = -8.93 and t = -9.73; P < or = 0.0005, respectively) than those observed for control female deer (1.22-1.38 fawns per female). During the second (0.36 fawns per female) and third summers (0.61 fawns per female) after the last booster injection, GnRH-treated female deer still produced significantly fewer fawns than did the controls (1.38 and 1.31 fawns per female, respectively). In one breeding season after treatment, five of 18 (28%) females vaccinated with PZP produced fawns, similar to the rate for GnRH-treated females (29%). In addition, females treated with GnRH had fewer oestrous cycles per female (0.06, P < or = 0.05) than did either control (0.22 cycles per female) or PZP-treated deer (0.36 cycles per female). Initial PZP treatment followed by a booster dose 5-7 months later reduced fawn production and prolonged the breeding season as females repeatedly returned to oestrus, similar to results reported in other studies.

Identification of subpopulations of bovine mammary-gland phagocytes and evaluation of sensitivity and specificity of morphologic and functional indicators of bovine mastitis.

The number and function of bovine mammary-gland phagocytes were assessed in 8 lactating cows, each tested at least twice within an 8-mo period (total number of observations, 20). Macrophages and polymorphonuclear (PMN) cells were evaluated by conventional cytology, flow cytometry, fluorescent microscopy, and somatic-cell count (SCC). Phagocytosis was evaluated from the uptake of fluorescent beads and expressed as median fluorescence intensity (MFI). Two major subpopulations of phagocytes, of low or high MFI (LFI or HFI), were observed, and there were up to 4 sub-subpopulations within the HFI subpopulation of both macrophages and PMN cells. Fluorescent microscopy identified phagocytes containing up to 4 beads per cell. Cows showing < or = 72.3% phagocytes by cytology were regarded as non-mastitic (11 observations), and those showing > or = 80.7% phagocytes were considered to be mastitic (8 observations). Phagocyte MFI was negatively associated with mastitis; that is, the higher the MFI, the lower the SCC. The percentage of HFI PMN cells was the only indicator of mastitis with 100% sensitivity and specificity. Thus, bovine mammary-gland phagocytes consist of several subpopulations of different phagocytic ability, whose assessment more adequately predicts bovine mastitis than do morphologic indicators.

Blood and milk cellular immune responses of mastitic non-periparturient cows inoculated with Staphylococcus aureus.

Lymphocyte function and phenotype of peripheral blood and mammary gland cells were evaluated in non-periparturient cows before and at 1, 4 to 8 and 9 to 14 d after inoculation with Staphylococcus aureus, as expressed by percentage of CD3+, CD2+, and CD45R+ cells, antigen density of these markers per lymphocyte, and mitogen-induced blastogenesis. Milk bacterial counts and somatic cell counts (SCC) were also assessed. Mitogen-induced blastogenic responses were strong in blood and weak in mammary gland cells in all observations and positively correlated with the percent of CD45R+ cells. Significantly greater percentages of milk CD3+ lymphocytes and increased CD3, CD2, and CD45R antigen density per cell were observed after challenge. The blood CD3 and CD2 antigen density per lymphocyte and the milk CD2+ lymphocyte percent were negatively correlated with SCC (P < or = 0.01). No mastitis (SCC < or = 500 000 cells/mL) was observed in cows showing blood lymphocyte CD2 and CD3 antigen density indices < or = 2.5 and 6, respectively. Forty-one percent of SCC values were predicted by the combined blood CD2 and milk CD3 antigen density (P < or = 0.01). These findings support the hypotheses that mitogen-induced lymphocyte blastogenesis is not a valid test to assess mammary gland immunocompetence and that CD2 expression may facilitate immune responses by decreasing the number of T cell receptors required to achieve full activation.

Longitudinal evaluation of bovine mammary gland health status by somatic cell counting, flow cytometry, and cytology.

Bovine mastitis phases induced by Staphylococcus aureus were assessed in 6 lactating cows before challenge and at 1, 4-8, and 9-14 days postinoculation (dpi). Milk lymphocytes, macrophages, and polymorphonuclear cells (PMN) were counted by conventional (manual) cytology, identified by CD3+ and CD11b+ immunofluorescence and counted by flow cytometry (based on leukocyte forward and side light scatter values). Somatic cell counts (SCC) and recovery of bacteria were recorded at the same times. Preinoculation samples showed a lymphocyte-dominated composition. At 1 dpi, the percentage of PMN increased and that of lymphocytes decreased. At 4-8 dpi, PMN were predominant, but the percentage of mononuclear cells increased above that at 1 dpi and further increased by 9-14 dpi (when lymphocytes approached prechallenge values). Based on leukocyte percentages, 3 indices were created from the data: 1) the PMN/lymphocyte percentage ratio (PMN/L), 2) the PMN/macrophage percentage ratio (PMN/M), and 3) the phagocyte (PMN and macrophage)/lymphocyte percentage ratio (Phago/L). Significant correlations were found between cytologic and flow cytometric data in all of these indicators (all with P < or = 0.01). These indices identified nonmastitic, early inflammatory (1-8 dpi), and late inflammatory (9-14 dpi) animals. In contrast, SCC and bacteriology did not. Although sensitivity of the SCC was similar to that of Phago/L, the specificity of SCC was almost half that of the Phago/L index. Based on flow cytometry indicators, an algorithm for presumptive diagnosis of bovine mastitis was developed. Flow cytometry provides results as valid as those obtained by conventional (manual) cytology, shows greater ability to identify mastitic cases than does SCC, and may identify 3 mammary gland health-related conditions.

Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants.

OBJECTIVES: To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. SAMPLE POPULATION: 39 human and 56 ruminant P aeruginosa isolates. PROCEDURES: Isolates were identified by use of bacteriologic techniques and automated Pvull-based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). RESULTS: All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of single-host ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. CONCLUSIONS AND CLINICAL RELEVANCE: Automated ribotyping with Pvull discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes.

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis.

OBJECTIVES: To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. ANIMALS: 10 first-lactation Holstein cows. PROCEDURE: Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. RESULTS: Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.

Longitudinal evaluation of CD4+ and CD8+ peripheral blood and mammary gland lymphocytes in cows experimentally inoculated with Staphylococcus aureus.

Staphylococcus aureus is a major pathogen associated with mastitis, a disease affecting both women and dairy cows. The longitudinal profiles of bovine peripheral blood and mammary gland lymphocyte phenotypes in response to S. aureus-induced mastitis were investigated in dairy cows. Increased percentage of CD4 lymphocytes in the mammary gland between 1 and 8 days post-inoculation, increased milk CD4 protein density per cell between 1-8 days post-inoculation, and a statistically significant negative correlation between post-inoculation bacterial counts in milk and blood lymphocyte CD4 protein density were found. Together with blood and milk leukocyte counts, the milk lymphocyte CD4/CD8 ratio and the milk lymphocyte CD4 protein density were more informative indicators than milk somatic cell counts and bacteriology for identification of early vs. late inflammatory phases. These findings suggest that CD4+ lymphocytes play a protective role in the early stages of S. aureus-induced mastitis.

Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks.

Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferi-infected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic culture, polymerase chain reaction (PCR) for bacterial DNA, immunologic responses, and gross lesions and histopathologic changes were investigated during the experiment or at necropsy 9 months after tick exposure. In all of the seven challenged ponies, infection with B. burgdorferi was detected from monthly skin biopsies and various tissues at postmortem examination by culture and by PCR. However, pony No. 178 exposed to laboratory-reared nymphs (without B. burgdorferi infection) and challenged with B. burgdorferi-infected adult ticks 2 months later did not develop a B. burgdorferi infection. All of the infected ponies seroconverted. Control ponies and pony No. 178 were negative by culture, PCR, and serology. Except for skin lesions, we failed to induce any significant histopathologic changes in this study. This is the first report of successful tick-induced experimental infection in ponies by exposure to B. burgdorferi-infected ticks. This Lyme disease model will be very useful to evaluate efficacy of vaccines against the Lyme agent and the effect of antibiotic therapy on horses infected with B. burgdorferi.

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450.

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. The compound phenanthrene is a model PAH. A novel fluorometric method for measuring phenanthrene metabolism in vitro was developed and verified with direct measurement of [14C]phenanthrene using dog liver microsomes. The fluorometric assay and direct measurement of [14C]phenanthrene metabolism were used to show that CYP6D1, a house fly cytochrome P450, is the major house fly P450 involved in phenanthrene metabolism. Phenanthrene was metabolized by microsomes from the LPR strain of house fly that overexpresses CYP6D1, but metabolism was not observed in the CS strain that has a lower level of CYP6D1. Furthermore, the majority of phenanthrene metabolism was inhibited by a CYP6D1-specific antibody. This study increases the number of known substrates of CYP6D1 and identifies polyaromatic hydrocarbons as potential substrates of CYP6D1. The utility of CYP6D1 as an agent in bioremediation and the utility of the new fluorometric assay for understanding PAH metabolism in insects and mammals are discussed.

Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses.

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.

A chimeric aryl hydrocarbon receptor knockout mouse model indicates that aryl hydrocarbon receptor activation in hematopoietic cells contributes to the hepatic lesions induced by 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

Pathologic changes associated with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been reported in the livers of a wide range of species. While these changes have been extensively described, the mechanisms of toxic interaction(s) that produce these lesions remain unclear. Using an aryl hydrocarbon receptor (Ahr) knockout male mouse chimeric model, we investigated whether the presence of this receptor in hematopoietic and/or parenchymal cells affects TCDD-induced hepatotoxicity. Bone marrow chimeras were produced by hematopoietic reconstitution of irradiated mice. Specifically, chimeras were generated with aryl hydrocarbon receptor (AHR) positive hematopoietic and parenchymal cells (Ahr+/+ animal bone marrow cells into irradiated Ahr+/+ animals), AHR positive hematopoietic and negative parenchymal cells (Ahr+/+ into Ahr-/-), AHR negative hematopoietic and positive parenchymal cells (Ahr-/- into Ahr+/+), and AHR negative hematopoietic and parenchymal cells (Ahr-/- into Ahr-/-). Male wild-type (Ahr+/+) and knockout (Ahr-/-) animals were used as nonchimeric controls. Following TCDD treatment (30 microg/kg body wt), liver sections from mice in each control and chimeric group were histologically evaluated for necrotic and inflammatory changes. TCDD treatment produced moderate inflammation in Ahr+/+ controls and Ahr+/+ into Ahr+/+ chimeras. This response was mild in TCDD-treated Ahr-/-, Ahr-/- into Ahr-/-, Ahr+/+ into Ahr-/-, and Ahr-/- into Ahr+/+ animals and was not different from the corresponding vehicle-treated groups. Moderate necrosis was observed in all TCDD-treated controls or chimeras with AHR-positive parenchyma. No or mild necrosis was observed in TCDD- and vehicle-treated animals containing AHR-negative parenchyma. These data indicate that the presence of AHR in hepatic parenchyma alone is sufficient for TCDD induction of hepatic necrosis, and its presence in hematopoietic cells is necessary for the inflammatory response to TCDD-induced hepatic lesions.

Induction of hepatic cytochromes P450 in dogs exposed to a chronic low dose of polychlorinated biphenyls.

Induction of cytochrome P450 isoforms, specifically CYP1A1, and their catalytic activities are potential biomarkers of environmental contamination by polychlorinated biphenyls (PCBs). In this study, dogs were exposed to 25 ppm or 5 ppm Aroclor 1248 (PCB mixture) daily in their diet for 10 or 20 weeks, respectively. Relative to controls, hepatic microsomes from dogs dosed with PCBs had higher levels of CYP1A1 detected in immunoblots and higher levels of EROD activity, but low levels of induction for CYP2B and PROD activity. Concentrations of 96 PCB congeners in serum and liver were evaluated using capillary chromatography. Results showed that all dogs exposed to PCB mixtures had higher levels of PCB in serum and liver. Dogs preferentially sequestered highly chlorinated PCB congeners in liver relative to serum. With these experiments, we demonstrated that EROD activity was a potentially sensitive marker of PCB exposure at 5 and 25 ppm. Furthermore, CYP1A1 and EROD activity were maximally induced in dogs consuming dietary concentrations only 2.5 times the maximal permissible level for human food (FDA). The value of CYP1A1 induction as a biomarker of PCB exposure was tenuous because neither CYP1A1 levels nor EROD activity correlated with total PCB body burden. However, a small subset of congeners were identified in liver that may strongly influence EROD and PROD induction. Finally, two dogs in the 25 ppm dose group were fasted for 48 h. After 24 h of fasting, several new congeners appeared in the serum and remained in the serum for the remainder of the fast. The fast caused a 293% increase in PCB concentration in serum. This increase has strong implications regarding mobilization of toxic PCBs in wildlife during fasting (e.g., migration, hibernation).

A primary immunodeficiency syndrome in Shar-Pei dogs.

A multiple immunodeficiency, involving antibody- and cell-mediated responses in 10 Chinese Shar-Pei (CSP) dogs is described. Abnormal levels of serum IgM and IgA in most cases, and IgG in fewer cases characterized the immunoglobulin deficiencies. Decreased in vitro proliferative responses of pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC) were found in nine cases. Clinical presentation involved several organ systems and was associated with recurrent infections and malignancy. Sera from affected dogs suppressed PWM-stimulated cell proliferation of affected and normal dogs, but not cultures stimulated with PWM followed by recombinant IL-2 (rIL-2). In vitro supplementation of PBMC cultures with immunomodulatory guanosine analogs (GA) resulted in increased de novo IgG and/or interleukin-6 (IL-6) synthesis. Cells from five immunodeficient dogs showed in vitro evidence of GA- or rIL-2-dependent enhanced immunological responses. Since rIL-2-mediated activation of the IL-2 receptor and GA-mediated immunomodulation are reported to act through protein kinase C (PKC)-independent pathways, it is concluded that the IL-2 receptor is functional in these dogs and that cell activation through alternative pathways may restore immune responses in affected CSP dogs.

Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells.

The inter-species cross-reactivity of cytokine bioassays for interleukin-2 (IL2) and interleukin-6 (IL6) was investigated in the canine species. The kinetics of normal canine peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM), were analyzed in terms of cytokine release and responsiveness to cytokine stimulation, in conjunction with determination of cell proliferation, de novo antibody synthesis and cell surface phenotype. PBMC were stimulated with PWM at the beginning of the culture and human recombinant IL2 (rIL2) was added 3-4 days post stimulation (d.p.s.). Mitogenically stimulated cells proliferated and synthesized antibody in a linear fashion up to 6 d.p.s. Resting PBMC had a mean CD4+/CD8+ ratio of 1.7:1; whereas cells stimulated with PWM were predominantly of CD8 phenotype at 7 d.p.s.. Three days after addition of IL2, stimulated cells were predominantly of the Thy+, sIg-, CD4+, CD8- phenotype, with an increase in the CD4+/CD8+ ratio. The magnitude of de novo antibody synthesis was lower in rIL2-supplemented cultures than in cultures stimulated only with PWM, and suggested a negative relationship between de novo antibody synthesis and proliferative responses of the same cultures. Supernatants from mitogen-stimulated cultures induced proliferation of mouse IL2- and IL6-dependent cell lines. Antibodies reactive with human IL2 or IL6 inhibited these responses. IL2-like activity in PWM-stimulated culture peaked by 2 d.p.s. and decreased thereafter. IL6-like activity peaked later (4-6 d.p.s.).

The pathogenesis of experimental toxic shock syndrome: the role of interleukin-2 in the induction of hypotension and release of cytokines.

Toxic shock syndrome (TSS) is a multisystem disorder characterized by fever, hypotension, and involvement of three other organ systems. The etiologic agent is a toxigenic strain of Staphylococcus aureus which secretes the exotoxin, TSST-1. The toxin is a superantigen which stimulates the immune system to produce interleukin-1 (IL-1), interleukin-2, and tumor necrosis factor (TNF). We hypothesized that TSST-1 induces the release of IL-2 which in turn is either directly involved or acts via an additional mediator to produce hypotension. We submitted four pairs of normal anesthetized adult female baboons to intravenous boluses of TSST-1. One baboon in each pair received anti-IL-2 intravenously and anti-IL-2 receptor intrathyroidally 15 min prior to TSST-1. The other baboon received the same dose and placement of anti-sheep red blood cell antibody. Systolic and diastolic blood pressure was recorded continuously and mean arterial pressure was calculated and plotted. IL-1, IL-2, IL-6, and TNF were measured in serum at varying times before and after toxin administration. Systolic, diastolic, and mean arterial pressure were significantly lower in the sham-treated group versus the experimental (anti-IL-2/IL-2R) group (p < .05 for all variables). In addition no differences were seen in any of the measurements between experimentally treated baboons and those receiving no TSST-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Friendliness to humans and defensive aggression in cats: the influence of handling and paternity.

This study was undertaken to examine and reduce the stress and aggressiveness associated with fear of handling in laboratory cats (Felis sylvestris catus). Thirteen litters of kittens from a specific pathogen-free breeding colony were divided into three treatment groups: two were early weaned, removed from the colony and caged individually with or without handling up to 8 weeks of age, and the third served as a control group, removed from the colony just before testing. Behavior tests measuring degree of friendliness to humans and response to physical restraint were performed at ages 8, 12, 16, and 20 weeks. Serum cortisol concentrations were measured after each test. Results indicate that litter and sire influenced tractability but that handling or individual caging of kittens did not. Posttest serum cortisol concentrations were below normal adult levels in most kittens, including those reacting fearfully during testing and aggressively during restraint, and, therefore, are not a reliable indicator of stress in juvenile cats.