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.     

Immunogenicity of bovine parainfluenza type 3 virus proteins encapsulated in nanoparticle vaccines,following intranasal administration to mice

Stimulation of long lasting, protective immunity to respiratory viruses is often difficult to achieve with conventional respiratory vaccines. Polymeric nanoparticles, incorporating viral proteins have been shown to offer sustained release of antigen, with consequent prolongued stimulation of the respiratory immune system. In this paper the efficacy of two nanoparticle vaccines (poly-lactide-co-glycolide, PLGA; polymethylmethacrylate, PMMA), incorporating proteins of bovine parainfluenza type 3 virus (BPI-3) was investigated. As a preliminary to experiments in calves, it was considered essential to demonstrate immunogenicity of the experimental vaccine in mice. Mice immunised with PLGA nanoparticles, containing BPI-3 proteins, developed higher levels of virus-specific antibody than mice immunised with the PMMA vaccine or with soluble viral proteins alone. Immunoblotting using serum from the vaccinated mice, demonstrated strong reactions against the major BPI-3 proteins.     

Effect of dorsal hip loading,sedation, and general anesthesia on the dorsolateral subluxation score in dogs

OBJECTIVE: To determine whether dorsal loading of the pelvis and type of chemical restraint affected the dorsolateral subluxation (DLS) score of dog hips. STYDY DESIGN: In vivo testing of diagnostic method. ANIMALS: Labrador retrievers, Greyhounds, and crosses between both breeds (n = 119 dogs). METHODS: Dorsal load was applied to the hips through a strap that was placed over the hips, and the peak vertical and steady-state vertical, ground-reaction forces exerted on the stifles were measured in the DLS position. The DLS score was measured with their hips under dorsal load and compared with the DLS score without load. For 24 dogs, the DLS score was measured both under chemical restraint (medetomidine) and under general anesthesia. Wilcoxon-signed rank test and paired t test was used to compare effects of dorsal load and restraint method on the left and right DLS hip score, and P <.05 was considered significant. RESULTS: Both stifles together accepted median 15% body weight in the vertical plane in the unloaded steady state. Loading the hips increased the steady-state, vertical ground-reaction force to 34% body weight. At 8 months of age, loading significantly decreased the DLS score from 65% +/- 11% (mean +/- SD) to 60% +/- 12% for left hips and 66% +/- 11% to 63% +/- 13% for right hips. The left hip DLS score was affected by load more consistently than the right hip. In general, loading the hips decreased the DLS score in all breeds by 4% to 6% except for the greyhounds, in which the DLS score was unchanged by dorsal loading. When grouped by their DLS scores, load significantly decreased DLS scores in dogs with unloaded scores greater than 55% (nondysplastic), whereas DLS scores of dogs with unloaded scores between 55% and 45% (dysplastic) and less than 45% did not change significantly with load. Unloaded scores did not change significantly when compared under sedation versus general anesthesia. However, load significantly decreased the DLS score under general anesthesia for the left hip but not for the right hip. CONCLUSIONS: Less than half of the vertical ground-reaction force normally exerted by the hindlegs of a standing dog was sufficient to induce hip subluxation in the DLS position. Although dorsal loading approximately doubled the steady-state, vertical ground-reaction force, the decrease in the DLS score under load was never greater than 6%. Furthermore, the DLS scores of dogs most likely to be dysplastic (based on DLS scores <45%) were not affected by load, whereas load slightly decreased DLS cores of dogs with DLS scores greater than 55%. CLINICAL RELEVANCE: External influences can change the DLS score significantly, but the magnitude of change is unlikely to be of clinical importance, making the method useful as a field screening test for both dysplastic and nondysplastic hips in young dogs.     

Methemoglobinemia and eccentrocytosis in equine erythrocyte flavin adenine dinucleotide deficiency

This report describes erythrocyte biochemical findings in an adult Spanish mustang mare that exhibited persistent methemoglobinemia, eccentrocytosis, and pyknocytosis that were not related to the consumption or administration of an exogenous oxidant. The methemoglobinemia was attributed to a deficiency in cytochrome-b5 reductase (Cb5R) activity, and the eccentrocytes and pyknocytes were attributed to a marked deficiency in reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase (GR) activity that resulted in decreased reduced glutathione concentration within erythrocytes. The GR activity increased to a near-normal value after addition of flavin adenine dinucleotide (FAD) to the enzyme assay, indicating a deficiency of FAD in erythrocytes. The methemoglobinemia, eccentrocytosis, and pyknocytosis were attributed to deficiency of FAD in erythrocytes because the GR and Cb5R enzymes use FAD as a cofactor. This deficiency in FAD results from a defect in erythrocyte riboflavin metabolism, which has not been documented previously in animals.     

Susceptibility to IVM in a field strain of Haemonchus contortus subjected to four treatments in a closed sheep-goat flock in Kenya

Susceptibility to IVM (IVM) of “strain A” Haemonchus contortus which had been exposed to IVM four times over a 2-year period was compared to IVM susceptibility of “strain C” H. contortus which had no prior field exposure to IVM, by in vivo and in vitro methods. In vivo, the percentage reduction in faecal egg counts (FEC) and the total worm counts (TWC) were compared between control animals (lambs and kids) and animals treated with low dose IVM (20 μg/kg). In vitro susceptibility to IVM was evaluated by larval migration inhibition (LMI) after the two strains of H. contortus were exposed to different concentrations of IVM. The dose response, measured as the proportion of larvae inhibited from migrating, was used to estimate LD₅₀. Although differences in response to IVM in the in vivo determinations were not significant, “strain A” H. contortus had a significantly higher LD₅₀ than “strain C” in the LMI assay. Coincident with the conduct of the in vivo experiment, it was observed that “strain A” H. contortus established and survived better than “strain C” in the control lambs.     

Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small intestinal tracts of domestic kittens and lambs

This study examines the ability of Giardia duodenalis trophozoites, isolated from a wild bird, to colonize the intestinal tracts of companion animals (kittens) and domestic ruminants (lambs). Trophozoites colonized the intestinal tracts of intraduodenally inoculated animals as demonstrated by increasing parasite burdens within the duodenum and jejunum and by fecal passage of cysts within 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated in kittens. In these animals, infection significantly reduced jejunal brush border microvillous length and density, which resulted in a loss of overall epithelial brush border surface area. This injury was associated with the production of diarrhea in four of five infected kittens. These findings indicate that some bird species may carry G. duodenalis that represent a possible health threat to companion animals and livestock. Our results describe the first successful colonization of avian-derived G. duodenalis trophozoites in the small intestines of domestic kittens and lambs.     

Traumatic body wall herniation in 36 dogs and cats

Traumatic body wall hernias (TBWH) are serious sequelae to traumatic injury in dogs and cats. During the study period, 26 dogs and 10 cats with surgically managed TBWH were identified. Five cases (four dogs, one cat) did not have their hernias identified during the first 24 hours of hospitalization. Bite wounds were the most common cause of TBWH, accounting for 54% of canine and 40% of feline hernias. Twelve cases (nine dogs, three cats) had serious intra-abdominal injuries diagnosed in addition to their TBWH. Seventy-three percent of dogs and 80% of cats survived to hospital discharge. In addition, the authors report the occurrence of a unique cause of herniation, termed an autopenetrating hernia.