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.     

Experimental infection with low and high pathogenicity H7N3 Chilean avian influenza viruses in Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera)

Two different wild duck species common in Chile and neighboring countries, Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera), were intranasally inoculated with 10(6) mean embryo infective dose (EID50) of the H7N3 low pathogenicity (LP) avian influenza virus (AIV) (A/chicken/Chile/176822/02) or high pathogenicity (HP) AIV (A/chicken/Chile/ 184240-1/02), in order to study the infectivity and pathobiology of these viruses. None of the virus-inoculated ducks had clinical signs or died, but most seroconverted by 14 days postinoculation (DPI), indicating a productive virus infection. Both LPAIV and HPAIV were isolated from oral swabs from two of six Chiloe wigeons and from oral and/or cloacal swabs from all five of the cinnamon teal at 2 DPI. Both LPAIV and HPAIV were efficiently transmitted to cinnamon teal contacts but not to Chiloe wigeon contacts. This study demonstrates that the cinnamon teal and Chiloe wigeons were susceptible to infection with both Chilean H7N3 LPAIV and HPAIV, but only the cinnamon teal showed contact transmission of the virus between birds, suggesting that the cinnamon teal has the potential to be a reservoir for these viruses, especially the LPAIV, as was demonstrated in 2001 with isolation of a genetically related H7N3 LPAIV strain in a cinnamon teal in Bolivia. However, the definitive source of the H7N3 Chilean LPAIV still remains unknown.     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

Comparative short-term efficacy of Oridermyl? auricular ointment and Revolution? selamectin spot-on against feline Otodectes cynotis and its associated secondary otitis externa

The efficacy of Oridermyl^® (acaricidal/antibiotic/antifungal/anti-inflammatory ointment) and Revolution^® (selamectin spot-on) was compared in a blinded randomized study on 24 adult cats with otoacariasis. Twelve cats were treated once daily for 10 d with Oridermyl^® and 12 cats were treated on Day 0 with Revolution^®. Otitis was assessed with cytological counts of mean number of bacteria and yeast on Days 0 and 10, and scored clinically every other day. All auricular secretions were removed for mite count on Day 10. On Day 0, cytological examination confirmed the presence of secondary bacterial (24/24) and fungal (21/24) infections. No live mites were observed otoscopically after Day 4 and in auricular secretions at Day 10 in both groups. On Day 10, secondary infections were resolved for all cats treated with Oridermyl^® but were present in all cats treated with Revolution^®. Improvement in clinical signs of otitis over time was superior in the Oridermyl^® group (P < 0.001).     

Effect of sample volume size and sampling method on feline longitudinal myocardial velocity profiles from color tissue Doppler imaging

ObjectivesThe aims of this study were to compare the effect of sample volume (SV) size settings and sampling method on measurement variability and peak systolic (s′), and early (e′) and late (a′) diastolic longitudinal myocardial velocities using color tissue Doppler imaging (cTDI) in cats.AnimalsTwenty cats with normal echocardiograms and 20 cats with hypertrophic cardiomyopathy.MethodsWe quantified and compared empirical variance and average absolute values of s′, e′ and a′ for three cardiac cycles using eight different SV settings (length 1,2,3 and 5 mm; width 1 and 2 mm) and three methods of sampling (end-diastolic sampling with manual tracking of the SV, end-systolic sampling without tracking, and random-frame sampling without tracking).ResultsNo significant difference in empirical variance could be demonstrated between most of the tested SVs. However, the two settings with a length of 1 mm resulted in a significantly higher variance compared with all settings where the SV length exceeded 2 mm (p < 0.001). There was an overall significant effect of sampling method on the variability of measurements (p = 0.003) and manual tracking obtained the lowest variance. No difference in average values of s′, e′ or a′ could be found between any of the SV settings or sampling methods.ConclusionWithin the tested range of SV settings, an SV length of 1 mm resulted in higher measurement variability compared with an SV length of 3 and 5 mm, and should therefore be avoided. Manual tracking of the sample volume is recommended.     

URETEROLITHIASIS AND PAPILLOMA FORMATION IN THE URETER OF A GUINEA PIG

A 5-year-old guinea pig with suspected urolithiasis was presented for radiology and ultrasound examinations of the abdomen. Radiographically, an irregular-shaped mineral opacity was detected in the area of the urinary bladder. Ultrasonographically, pyelectasia of the right kidney, hydroureter with an ureterolith cranial to a thickened ureter wall close to the ureterovesical junction, and a thickened urinary bladder wall were detected. Histopathologically, the thickened ureter wall was found to be a papilloma. The ureter calculus consisted of 100% calcite.     

Isolation and identification of Mycobacterium kansasii from pleural fluid of a dog with persistent pleural effusion

A 3-year-old spayed female Whippet was examined for cough and respiratory distress. Lung lobe torsion with pleural effusion was diagnosed, and lung lobectomy was performed. Pleural effusion recurred during the following 27 months; conventional bacteriologic cultures of pleural effusion did not result in bacterial growth. A second lung lobectomy, pleuroperitoneal shunt placement. and pericardectomy were subsequently performed. Mycobacterium kansasii was eventually isolated from pleural fluid and identified by polymerase chain reaction amplification and DNA sequencing. The dog was euthanatized before therapeutic response could be evaluated. To our knowledge, this is the first report of M. kansasii infection in a dog. Additionally, this is the first report of mycobacterial isolation from pleural fluid, and one of few reports of antemortem mycobacterial isolation from a body fluid, as opposed to identification in specimens during histologic examination. Routine bacteriologic culture methods are insufficient to isolate mycobacterial agents, and special methods are indicated in dogs with persistent pleural effusion.     

Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.     

Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.     

Babesia odocoilei infection in elk

Two male North American elk from a commercial herd were evaluated because of a sudden onset of lethargy, anorexia, and voiding of red urine. These 2 elk were kept in the same pen as 4 other male elk that had died during the preceding 2 months. Laboratory analyses revealed anemia and intraerythrocytic parasites, later confirmed as Babesia odocoilei (a protozoal hemoparasite of cervids). Of the 240 elk remaining in the herd, 59 were screened for B odocoilei by microscopic evaluation of blood smears, protozoal culture of blood, and immunofluorescent antibody testing of serum. Of those 59 elk, 34 (58%) were infected with B odocoilei. Babesia odocoilei infection in elk can be fatal and should be considered in cases of sudden death or acute hemolytic anemia. Familiarity with the disease in elk is essential for practitioners because of the increasing popularity of commercial elk farming.