Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

Accuracy of heart rate obtained by auscultation in atrial fibrillation

The accuracy of heart rate estimation by cardiac auscultation over a 15-second period, and the influence of clinical experience on accuracy were evaluated in a dog with chronic atrial fibrillation by test subjects of varying experience. Only 30% of all test subjects provided accurate heart rate estimates. Board-certified specialists, medicine residents, and experienced nurses were significantly more accurate in their estimates than surgery residents and students. Accurate estimates were provided by 12.5% of surgery residents and students, as opposed to 64% of the other test subjects. Auscultatory estimates of heart rate in atrial fibrillation may be significantly inaccurate, and under some circumstances they may not provide a sound basis for making clinical decisions.     

Effect of administration of a phospholipid emulsion on the initial response of horses administered endotoxin

OBJECTIVE: To evaluate the effect of a phospholipid emulsion (PLE) on the initial response of horses to administration of endotoxin. ANIMALS: 12 healthy adult horses. PROCEDURES: Horses were assigned to 2 treatment groups (6 horses/group). The control group was administered 1 L of saline (0.9% NaCl) solution, and the treated group was administered PLE (200 mg/kg, IV); treatments were administered during a period of 120 minutes. An infusion of endotoxin was initiated in both groups starting 1 hour after initiation of the saline or PLE solutions. Physical examination and hemodynamic variables were recorded, and blood samples were analyzed for concentrations of tumor necrosis factor (TNF)-alpha, interleukin-6, thromboxane B2 (TxB2), 6 keto-prostaglandin F (PGF)1alpha, total leukocyte count, and PLE concentrations. An ANOVA was used to detect significant differences. RESULTS: Administration of PLE resulted in significantly lower rectal temperature, heart rate, cardiac output, right atrial pressure, and pulmonary artery pressure and higher total leukocyte counts in treated horses, compared with values for control horses. The TNF-alpha concentration was significantly less in treated horses than in control horses. The TxB2 and 6 keto-PGFF1alpha concentrations were significantly different between treated and control horses at 30 minutes (TxB2) and at 30 and 60 minutes (6 keto-PGF1alpha). CONCLUSIONS AND CLINICAL RELEVANCE: Prior infusion of PLE in horses administered a low dose of endotoxin decreased rectal temperature, heart rate, pulmonary artery pressure, and TNF-alpha concentrations. Results of this study support further evaluation of PLE for use in the treatment of horses with endotoxemia.     

Sensitivity of test strategies used in the Voluntary Johne's Disease Herd Status Program for detection of Mycobacterium paratuberculosis infection in dairy cattle herds

OBJECTIVE: To evaluate sensitivities at the herd level of test strategies used in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and alternative test strategies for detecting dairy cattle herds infected with Mycobacterium paratuberculosis. DESIGN: Nonrandom cross-sectional study. SAMPLE POPULATION: 64 dairy herds from Pennsylvania, Minnesota, Colorado, Ohio, and Wisconsin. Fifty-six herds had at least 1 cow shedding M. paratuberculosis in feces; the other 8 herds were free from paratuberculosis. PROCEDURE: For all adult cows in each herd, serum samples were tested for antibodies to M. paratuberculosis with an ELISA, and fecal samples were submitted for bacterial culture for M. paratuberculosis. Sensitivities at the herd level (probability of detecting infected herd) of various testing strategies were then evaluated. RESULTS: Sensitivity at the herd level of the testing strategy used in level 1 of the VJDHSP (use of the ELISA to test samples from 30 cows followed by confirmatory bacterial culture of feces from cows with positive ELISA result) ranged from 33 to 84% for infected herds, depending on percentage of cows in the herd with positive bacterial culture results. If follow-up bacterial culture was not used to confirm positive ELISA results, sensitivity ranged from 70 to 93%, but probability of identifying uninfected herds as infected was 89%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the testing strategy used in the VJDHSP will fail to identify as infected most dairy herds with a low prevalence of paratuberculosis. A higher percentage of infected herds was detected if follow-up bacterial culture was not used, but this test strategy was associated with a high probability of misclassifying uninfected herds.     

Marek's disease in turkeys. II. Characterization of the viral glycoprotein B gene and antigen of a turkey strain of Marek's disease virus

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.     

A comparison of catecholamine and cortisol responses of young lambs and calves to painful husbandry procedures

AIM: To compare the changes in plasma concentrations of noradrenaline, adrenaline and cortisol in lambs after ring castration plus tailing and in calves after dehorning with or without prior local anaesthesia. METHODS: Male lambs were castrated and tailed with rings and calves were dehorned by amputation using a scoop with or without prior local anaesthesia. Blood samples were taken before and after treatment and plasma concentrations of noradrenaline, adrenaline and cortisol were determined. RESULTS: Castration plus tailing of lambs resulted in a rapid increase in noradrenaline concentrations, a lack of an adrenaline response and a marked increase in cortisol concentration. There were similar changes in catecholamine concentrations in calves that were dehorned both with and without local anaesthetic, with adrenaline being elevated within 5 min of treatment and noradrenaline exhibiting a more protracted response. Dehorning caused a marked cortisol increase which was reduced to control concentrations by local anaesthesia for as long as the associated nerve blockade lasted. CONCLUSIONS: The very short-lived adrenaline responses in calves were attributed to dehorning-induced nociceptor input leading to sympathetic stimulation of the adrenal medulla. The longer lasting noradrenaline responses in lambs and calves were thought be due to 'wash-out' of noradrenaline from damaged tissue associated with rings and amputation wounds, respectively.