Chester B. Thomas

ADHERENCE TO BOVINE NEUTROPHILS AND SUPPRESSION OF NEUTROPHIL CHEMILUMINESCENCE BY MYCOPLASMA BOVIS

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.

Comparative diagnostic test characteristics of oscillometric and Doppler ultrasonographic methods in the detection of systolic hypertension in dogs.

Comparison of test characteristics allows a clinician to choose the optimal diagnostic test method for an individual patient. This study assessed the comparative test characteristics of noninvasive (NI) blood pressure measurement methods (oscillometric and Doppler) and used this information to develop optimal cutoff values for diagnosis of systolic hypertension in dogs by these NI methods. Simultaneous NI (oscillometric or Doppler methods) and invasive (arterial puncture [AP]) systolic blood pressure (SBP) measurements were obtained prospectively from normal dogs and dogs suspected of having systemic hypertension based on clinical signs. Oscillometric SBP readings were obtained from the distal hind limb (Osc-L, n = 54) or the proximal tail (T. n = 27). Doppler BP measurements were obtained using a forelimb cuff (n = 57). AP-SBP was categorized as hypertensive if > or = 160 mmHg, and sensitivity (Se). specificity (Sp), and likelihood ratios (LR) were calculated for diagnostic cutoff values ranging from 130 to 220 mmHg. Receiver operator characteristic (ROC) curves were analyzed to determine optimal cutoff values for diagnosis of AP-SBP > or = 160 mmHg. Optimal NI SBP cutoff values considered to reflect AP values > or = 160 mmHg were: Osc-L = 160 mmHg (Se: 65%, Sp: 85%. LR = 4.33: 1), Osc-T = 150 mmHg (Se: 84%, Sp: 75%, LR = 3.36: 1), and Doppler = 160 mmHg (Se: 71%,

Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.

Mycoplasma bovis suppression of bovine lymphocyte response to phytohemagglutinin.

The effect of viable Mycoplasma bovis on the in vitro bovine peripheral blood lymphocyte response to phytohemagglutinin (PHA) was studied. Results showed that M. bovis did not act as a mitogen for bovine lymphocytes. Viable M. bovis produced a dose and time dependent suppression of the PHA stimulated lymphocyte response. Suppression was not a result of differences in the viability of infected or control lymphocyte cultures. The suppressive effect of M. bovis was found to be independent of the concentration of PHA used in the test and the lymphocyte response could not be restored by supplementation of the culture medium with arginine. Delay for 48 h after PHA stimulation before adding M. bovis to the lymphocyte cultures diminished, but did not prevent, the suppression of the lymphocyte response. These results show that suppression of the lymphocyte response does not require the presence of M. bovis during the period of PHA stimulation, and that M. bovis was capable of interrupting [3H]-thymidine incorporation in lymphocytes which were actively synthesizing DNA.

Effect of milk sample collection strategy on the sensitivity and specificity of bacteriologic culture and somatic cell count for detection of Staphylococcus aureus intramammary infection in dairy cattle

Four milk sample collection strategies for bacteriologic culture and identification of bovine intramammary infection due to Staphylococcus aureus were evaluated. Milk samples were collected at 24 h intervals from 245 lactating mammary quarters of 62 cows from one commercial dairy herd on 6 successive days. A total of 1470 quarter milk samples were available for study. Based on the bacteriologic culture results of all six quarter milk samples, each quarter was classified as infected with or free of S. aureus. The case definition used to establish the ‘gold standard’ was the isolation of two or more colonies of S. aureus on two or more occasions from the six quarter milk samples obtained from a given mammary quarter. The probability of a false-negative classification of a mammary quarter using all six culture results was estimated to be less than 0.0046, while the probability of a false-positive classification was less than 0.0004. Twenty-two quarters from 16 of 62 cows had S. aureus intramammary infection. Inocula (0.1 ml) for bacteriologic culture were prepared in the laboratory from quarter milk samples to represent alternative strategies for milk sample collection on farms. Sensitivity and specificity of detection of S. aureus-infected mammary quarters and/or cows was then determined. The accuracy of somatic cell counts for the same purpose was also determined for several cut-off values. The range of sensitivity values for bacteriologic culture and SCC were 91–100% and 54–95%, respectively. The range of specificity values for each test method ranged from 97.6 to 100% and from 81 to 83%, respectively. Bacteriologic culture, using any of the sampling strategies examined, had high specificity ( > 98%) and relatively high sensitivity ( > 91%) for identifying S. aureus intra mammary infection (IMI). However, there was a great difference in the number of culture attempts necessary to achieve this accuracy which would influence a dairy farm managers choice of which type of milk sample collection strategy to use.

A linear programming assessment of the profit from strategies to reduce the prevalence of Staphylococcus aureus mastitis

We used a linear programming model to estimate the financial returns to a Staphylococcus aureus testing and control program over a 1-year period for a 100-cow herd, with a 8636-kg rolling-herd average. Six tests, which vary in sensitivity from 0.80 to 0.98 and specificity of 0.99, were examined in simulated herds with 10, 20 and 30% prevalence of S. aureus infection. Sensitivity of these results to a range of assumptions regarding rolling-herd average, milk price, somatic cell-count premium, and cost and cure rate of dry treatment were examined to determine the profits from the program. The profits of a control program are most dependent upon prevalence, cell-count premium, and cost of dry treatment. In our simulation for a 100-cow herd, a testing and control program appears to cost less than US

Evaluation of Serum Ferritin as a Tumor Marker for Canine Histiocytic Sarcoma

10 per cow per year, and pays for itself within 1 yr, except under the lowest prevalence and most-adverse conditions (low yield, high cost of dry treatment, or low SCC premium.

A model to determine sampling strategies and milk inoculum volume for detection of intramammary Staphylococcus aureus infections in dairy cattle by bacteriological culture

BACKGROUND Canine histiocytic sarcoma (HS) is an aggressive malignancy. Hyperferritinemia has been documented in dogs with HS and could serve as a tumor marker aiding in diagnosis and treatment. In people, hyperferritinemia is found in inflammatory diseases, liver disease, and hemolysis, and thus may occur in dogs with these conditions. OBJECTIVE To determine if serum ferritin concentration is a tumor marker for canine HS. ANIMALS Dogs with HS (18), inflammatory diseases (20), liver disease (24), immune-mediated hemolytic anemia (IMHA) (15), and lymphoma (23). METHODS Prospective, observational, cohort study: Serum ferritin concentration was measured at initial diagnosis. Parametric methods were used to compare mean log ferritin concentrations among disease categories. Receiver-operating characteristic curves and likelihood ratios were used to evaluate serum ferritin concentration as a tumor marker. RESULTS Varying proportions of dogs with IMHA (94%), HS (89%), liver disease (79%), lymphoma (65%), and inflammatory diseases (40%) had hyperferritinemia. Dogs with IMHA had significantly higher mean ferritin concentration than dogs in all other categories. Dogs with HS had significantly higher mean ferritin concentration than those in the inflammatory disease and lymphoma categories. Mean serum ferritin concentration was not significantly different between dogs with HS and those with liver disease. Decision thresholds were determined to distinguish IMHA and HS from the other diseases associated with hyperferritinemia. CONCLUSION Hyperferritinemia is common in dogs with HS and, after IMHA is ruled out, the degree of hyperferritinemia may be useful in differentiating dogs with HS from dogs with inflammatory diseases, liver disease, and lymphoma.

An application of sampling theory in animal disease prevalence survey design

Abstract A model was developed to evaluate the effects that methods of obtaining milk samples and culture inoculum volumes had on the sensitivity of microbiological culture to detect Staphylococcus aureus intramammary infections (IMI). An assumption was made that milk from mammary quarters infected with S. aureus only contains bacteria intermittently. A modified sine wave function was used to model this intermittent shedding pattern. Specifications for the components of the shedding cycle used in this function were based on quantitative culture results from 54 experimentally infected S. aureus quarters, sampled daily for a period of 30–49 days. The components of the shedding cycle were length in days, peak number of CFU shed per milliliter of milk, and length of time in the cycle when no shedding occurred. These components were used to estimate the models predicted distribution of S. aureus CFU ml −1 milk when individual quarter milk samples were cultured for S. aureus . The sensitivity of culture for several sampling methods was then calculated. The model predicted that culture of a single quarter milk sample had a sensitivity ranging from 60 to 87% for detection of S. aureus IMI depending on inoculum volume. Quarter milk samples taken on day 1 and repeated either on day 3 or day 4, and cultured separately using 0.1 ml of milk for culture inoculum, were predicted to have sensitivities of 90–95% and 94–99%, respectively. Other milk-sampling strategies examined included culture of a composite milk sample (equal-volume mixture of milk from four separate mammary quarters ) and pooled milk samples in which samples from different milkings (either quarter or composite samples) were mixed together and then cultured. The range of predicted sensitivities of these other sampling strategies was 30–97%. Factors having the greatest impact on the sensitivity of culture, in order of importance were: the type of milk sample, the volume of milk cultured, and the time interval between repeated milk sample collection strategies.

Development of an immunobinding assay with monoclonal antibodies to diagnose Mycoplasma bovis in semen.

Abstract The disease prevalence survey is one research approach available to epidemiologists working in preventive veterinary medicine. This paper demonstrates an application of sampling theory in the design of disease prevalence surveys and suggests ways in which this use of sampling methodology may enable the veterinary epidemiologists to better achieve their research objectives.