Streptococcus spp. were recovered from diseased tilapia in Thailand during 2009–2010 (n = 33), and were also continually collected from environmental samples (sediment and water) from tilapia farms for 9 months in 2011 (n = 25). The relative percent recovery of streptococci from environmental samples was 13–67%. All streptococcal isolates were identified as S. agalactiae (group B streptococci [GBS]) by a species-specific polymerase chain reaction. In molecular characterization assays, 4 genotypic categories comprised of 1) molecular serotypes, 2) the infB allele, 3) virulence gene profiling patterns (cylE, hylB, scpB, lmb, cspA, dltA, fbsA, fbsB, bibA, gap, and pili backbone–encoded genes), and 4) randomly amplified polymorphic DNA (RAPD) fingerprinting patterns, were used to describe the genotypic diversity of the GBS isolates. There was only 1 isolate identified as molecular serotype III, while the others were serotype Ia. Most GBS serotype Ia isolates had an identical infB allele and virulence gene profiling patterns, but a large diversity was established by RAPD analysis with diversity tending to be geographically dependent. Experimental infection of Nile tilapia (Oreochromis niloticus) revealed that the GBS serotype III isolate was nonpathogenic in the fish, while all 5 serotype Ia isolates (3 fish and 2 environmental isolates) were pathogenic, with a median lethal dose of 6.25–7.56 log10 colony-forming units. In conclusion, GBS isolates from tilapia farms in Thailand showed a large genetic diversity, which was associated with the geographical origins of the bacteria.
A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.
Intraparenchymal spinal cord tumors in the cat are rarely reported and often as single case reports. In the current study, the clinical, magnetic resonance imaging (MRI), histologic, and immunohistochemical features of 7 cases of intraparenchymal spinal cord tumors in the cat are described. All cats were domestic breed, ranged from 4 to 12 years of age (median 8 years), and included spayed females (5/7) and neutered males (2/7). The duration of clinical signs ranged from 2 weeks to 3 months. MRI revealed lesions that were hyperintense on T2-weighted images with variable contrast enhancement. All 7 tumors had histologic features consistent with glial origin: 3 were astrocytic (gemistocytic or fibrous), and 2 were oligoastrocytic. Single cases of oligodendroglioma and gliomatosis cerebri were also present in the study. Glial fibrillary acidic protein immunoreactivity was robust in the tumors that were predominately astrocytic, and the gliomatosis cerebri case had extensive BLA.36 and Iba1 immunoreactivity. Ki-67 immunoreactivity was variable and most abundant in the case of malignant oligoastrocytoma. The majority of peritumoral lymphocytes were CD3 positive. The current study expands upon the known reports of spinal cord neoplasia in the cat, confirms a caudal cervical segment predilection, and includes a report of gliomatosis cerebri in the spinal cord of a cat.
An unusual case of osteomyelitis caused by Nocardia cyriacigeorgica infection and resulting in mandibular osteomyelitis and cellulitis (lumpy jaw) is described in a young cat. A 1-cm hard nodular mass was an incidental finding in the right mandible of a 14-month-old cat during routine physical examination. The lesion was fast growing, reaching up to 6 cm in its largest dimension over a 5-week period. A core biopsy of the affected mandible revealed foci of osteolysis, woven bone formation, and a few large clusters of filamentous bacteria surrounded by fine eosinophilic amorphous material bordered by neutrophils, plasma cells, macrophages, and occasional multinucleated giant cells. Pure cultures of acid-fast variable, Gram-positive filamentous bacteria were recovered on blood and chocolate agar plates at 48-hr postinoculation. On amplification and sequencing of the 16S ribosomal RNA and 65-kDa heat shock protein genes, the microorganisms were identified as N. cyriacigeorgica, within the actinomycetes.
Brucella species infect a wide range of hosts with a broad spectrum of clinical manifestations. In mammals, one of the most significant consequences of Brucella infection is reproductive failure. There is evidence of Brucella exposure in many species of marine mammals, but the outcome of infection is often challenging to determine. The eastern Pacific stock of northern fur seals (NFSs, Callorhinus ursinus) has declined significantly, spawning research into potential causes for this trend, including investigation into reproductive health. The objective of the current study was to determine if NFSs on St. Paul Island, Alaska have evidence of Brucella exposure or infection. Archived DNA extracted from placentas (n = 119) and serum (n = 40) samples were available for testing by insertion sequence (IS) 711 polymerase chain reaction (PCR) and the Brucella microagglutination test (BMAT), respectively. As well, placental tissue was available for histologic examination. Six (5%) placentas were positive by PCR, and a single animal had severe placentitis. Multilocus variable number tandem repeat analysis profiles were highly clustered and closely related to other Brucella pinnipedialis isolates. A single animal was positive on BMAT, and 12 animals had titers within the borderline range; 1 borderline animal was positive by PCR on serum. The findings suggest that NFSs on the Pribilof Islands are exposed to Brucella and that the organism has the ability to cause severe placental disease. Given the population trend of the NFS, and the zoonotic nature of this pathogen, further investigation into the epidemiology of this disease is recommended.
Cutaneous meningiomas are rare tumors in human beings and animals. Two canine cases of cutaneous meningiomas affecting the eyelid are described in the current study: the first from a 5-week-old female Springer Spaniel dog with an 8 cm in diameter congenital mass expanding the left upper eyelid and medial canthus; the second from a 10-year-old female spayed Maltese–Poodle mix dog with 3 firm subcutaneous nodules affecting the right upper eyelid. All masses were removed surgically. Histologically, tumors were composed of spindle-to-epithelioid cells arranged in small lobules forming solid concentric whorls. Neoplastic cells were positive for vimentin and S100 and negative for pancytokeratin, glial fibrillar acid protein, and neurofilament. Transmission electron microscopy revealed meningothelial cells with convoluted interdigitating processes, desmosomes, and hemidesmosomes, and moderate numbers of cytoplasmic microfilaments. None of the cases presented a primary neuroaxial meningioma. The first case presents clinicopathological features consistent with human type I (congenital) cutaneous meningioma. The second case is consistent with a type II (acquired ectopic) tumor, and both are hypothesized to arise from ectopic arachnoid cells displaced during development.
Blood smears from a 24-year-old male rhesus macaque (Macaca mulatta) used for cognitive function studies were evaluated. The macaque had an 8-month history of gradual weight loss and increasing lymphocytosis. Most of the lymphocytes present were small to medium and had a mature morphology. Based on the degree and duration of the lymphocytosis, and the appearance of the lymphocytes, a diagnosis of chronic lymphocytic leukemia was made. The animal tested negative for 4 viral diseases that are commonly associated with lymphoproliferative disorders in Old World monkeys. Over the course of 12 months, the lymphocytosis progressed from 18.4 to 384 x 103 lymphocytes/µl (reference range: 0.8–17 x 103 cells/µl), and euthanasia was elected. On histologic examination, cluster of differentiation (CD)3- and CD8-positive, CD79-negative neoplastic cells comprised 40–60% of the bone marrow, diffusely obscured the normal splenic architecture, and were present in the vascular channels in other organs. Findings were characteristic of T-cell lymphocytic leukemia. Naturally occurring T-cell lymphocytic leukemia has been rarely reported in rhesus macaques and, to the authors’ knowledge, never in males. A persistent lymphocytosis characterized by a monomorphic population of CD3- and CD8-positive cytotoxic T-lymphocytes and the presence of neoplastic cells in the bone marrow led to a diagnosis in the current case.
A 22-month-old, female red deer (Cervus elaphus) was submitted to the University of Minnesota Veterinary Diagnostic Laboratory for necropsy and chronic wasting disease (CWD) testing. The deer was found positive for the abnormal prion protein in the obex and the retropharyngeal lymph node by immunohistochemical staining. Microscopic lesions of spongiform encephalopathy and immunohistochemical staining patterns and intensity were similar to those in CWD-positive elk and experimentally infected red deer.
Chlamydia spp., Coxiella burnetii, and Neospora caninum are responsible for reproductive diseases and are closely linked with high abortion rates in ruminants. Furthermore, C. burnetii and Chlamydia spp. have zoonotic potential. A real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of Chlamydia spp.,C. burnetii, and N. caninum. The detection of beta-actin as internal control in the same PCR reaction provides additional information about sample quality by detecting the presence of PCR inhibitors. The multiplex real-time PCR developed in the current study shows a greater sensitivity compared to previously used single-target PCR reactions with a reproducible detection limit of 0.13 plasmid copies per PCR for each target. Additional parallel amplification of all detectable pathogens did not adversely impact sensitivity. This new multiplex PCR allows the highly sensitive, cost-effective, and rapid detection of 3 important pathogens and has the potential to be a useful time-saving tool in the routine diagnosis of abortion cases in ruminants.
Brucella abortus RB51 is the vaccine strain currently licensed for immunizing cattle against brucellosis in the United States. Most cattle are vaccinated as heifer calves at 4–12 months of age. Adult cattle may be vaccinated in selected high-risk situations. Two herds of pregnant adult cattle in the brucellosis-endemic area of Wyoming were vaccinated with a standard label dose (1.0–3.4 x 1010 organisms) of RB51. Reproductive losses in the vaccinated herds were 5.3% (herd A) and 0.6% (herd B) and included abortions, stillbirths, premature calves, and unbred cows (presumed early abortion). Brucella abortus was cultured from multiple tissues of aborted and premature calves (7/9), and from placenta. Isolates were identified as B. abortus strain RB51 by standard strain typing procedures and a species-specific polymerase chain reaction. Bronchopneumonia with intralesional bacteria and placentitis were observed microscopically. There was no evidence of involvement of other infectious or toxic causes of abortion. Producers, veterinarians, and laboratory staff should be alert to the risk of abortion when pregnant cattle are vaccinated with RB51, to potential human exposure, and to the importance of distinguishing field from vaccinal strains of B. abortus.
In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods’ characteristics.
A 75.9-kg, 3.5-year-old male Irish Wolfhound dog with a 2–3-week history of gagging and eating difficulties was referred to the University of Florida Veterinary Medical Hospital (Gainesville, Florida) for evaluation of a large cranial mediastinal mass suspected to be a thymoma or lymphosarcoma. The patient had 4 months of nearly 10 kg progressive weight loss with severe flank sensitivity and radiographically apparent lumbar vertebral changes interpreted as discospondylitis. Lab work revealed hyperglobulinemia, mild proteinuria, normal T4, negative Brucella canis titer, and negative blood and urine bacterial cultures. A thoracotomy revealed a nonresectable, destructive, space-occupying mediastinal mass resulting in euthanasia without surgical recovery. Biopsies from the mass were collected during surgery for histology. Microscopic examination revealed extensive granulomatous cellulitis and lymphadenitis characterized by central cavitated necrotic areas containing debris and degenerate neutrophils, intermediate zones of fibrovascular proliferation with marked mixed inflammation, peripheral fibrosis, frequent multinucleated macrophages, and scattered mineralization. The necrotic material contained dense mats of 2 µm wide by 8–15 µm long fungal hyphae with parallel walls, acute angle branching, frequent septae, and occasional bulb-like dilations. DNA sequencing and phylogenetic analysis of the internal transcribed spacer region confirmed the presence of a fungus in the Inonotus tropicalis group. Inonotus tropicalis is primarily a wood decay fungus that is found on dead wood from angiosperms in tropical and subtropical habitats. Isolates of the I. tropicalis group have been detected a few times from immunosuppressed human beings with X-linked granulomatous disease.
Approximately 8,000 isolates of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli, isolated by 25 veterinary laboratories across North America between 2002 and 2010, were tested for in vitro susceptibility to beta-lactam, macrolide, and lincosamide drugs. The minimal inhibitory concentrations (MICs) of the beta-lactam drugs remained low against most of the Gram-positive strains tested, and no substantial changes in the MIC distributions were seen over time. Of the beta-lactam antimicrobial agents tested, only ceftiofur showed good in vitro activity against E. coli. The MICs of the macrolides and lincosamides also remained low against Gram-positive mastitis pathogens. While the MIC values given by 50% of isolates (MIC50) for erythromycin and pirlimycin and the streptococci were all low (≤0.5 µg/ml), the MIC values given by 90% of isolates (MIC90) were higher and more variable, but with no apparent increase over time. Staphylococcus aureus showed little change in erythromycin susceptibility over time, but there may be a small, numerical increase in pirlimycin MIC50 and MIC90 values. Overall, the results suggest that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to beta-lactam, macrolide, and lincosamide drugs tested over the 9 years of the study.
Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 x 102 copies of the positive control plasmid and 10–3 ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus–infected cats and showed a negative relationship (r = –0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.
A 9-year-old female spayed Domestic Medium Hair cat presented to the referring veterinarian with a 2-week history of sneezing, which progressed to swelling over the nasal planum. The cat had been under veterinary care for inflammatory bowel disease and had been treated with 1.25 mg/kg prednisolone once a day for approximately 1 year. On physical examination, an approximately 2–3 mm diameter, round polypoid pink soft-tissue mass was protruding slightly from the right nostril. Through histologic examination of representative sections from the mass, there was a severe diffuse infiltrate of epithelioid macrophages and neutrophils that surrounded frequent 15–20 µm yeast organisms. A Grocott methenamine silver stain revealed the presence of pseudohyphae in addition to the previously noted yeast forms. Real-time polymerase chain reaction (PCR) for Cryptococcus neoformans, Ajellomyces dermatitidis (syn. Blastomyces dermatitidis), Coccidioides immitis, Ajellomyces capsulatus (syn. Histoplasma capsulatum), Malassezia spp., and Candida spp. was performed on the paraffin-embedded sample. The PCR for Candida spp. was positive; the product was then sequenced and was determined to be consistent with Candida parapsilosis. Following the PCR diagnosis and prior to treatment of the infection, C. parapsilosis was cultured from a nasal swab. The infection in the cat in the current report was considered opportunistic and secondary to immunosuppression, following treatment for the inflammatory bowel disease.
Vesicular stomatitis is a viral disease primarily affecting horses and cattle when it occurs in the United States. Outbreaks in the southwestern United States occur sporadically, with initial cases typically occurring in Texas, New Mexico, or Arizona and subsequent cases occurring in a northward progression. The viruses causing vesicular stomatitis can be transmitted by direct contact of lesioned animals with other susceptible animals, but transmission is primarily through arthropod vectors. In 2012, an outbreak of vesicular stomatitis in the United States occurred that was caused by Vesicular stomatitis New Jersey virus serotype. Overall, 51 horses on 36 premises in 2 states were confirmed positive. Phylogenetic analysis of the virus indicated that it was most closely related to viruses detected in the state of Veracruz, Mexico, in 2000.
Haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), and white blood cells (WBC) were assessed in 20 dogs divided into 2 groups. The dogs of group A were not subjected to hunting exercise (control group), while the dogs of group B were subjected to hunting exercise for 3 hr (experimental group). Blood samples were collected from each animal before hunting (T0), immediately after 3 hr of hunting (T1), and after 1 hr of recovery (T2). The general linear model (GLM) repeated measures procedure showed a significant difference between the 2 groups (P < 0.0001) and a significant rise (P < 0.0001) in concentration of Hp, SAA, and CRP after hunting exercise, with a consequent decline during recovery period in group B. These parameters could be considered valid and easily obtainable biomarkers in relation to hunting stress in dogs. Additional studies will continue to elucidate the magnitude and the time of response of other acute phase proteins.
A 1-day-old female Holstein–Friesian calf was presented for severe dyspnea. Physical examination revealed respiratory distress, moderate edema of the ventral neck, and swollen jugular veins. The calf died and was submitted for necropsy. A severely enlarged thymus (40 cm x 20 cm x 10 cm) weighing 1.37 kg was detected on gross examination. Histomorphology was normal but no tingible body macrophages were observed in the medullary areas. Immunohistochemistry was characterized by the lack of thymic cluster of differentiation 3 and major histocompatibility complex class II expression compared to age-matched controls. The findings were consistent with severe thymic hyperplasia, a rare congenital condition that is also described in children. Immunohistochemical findings were suggestive of impaired T-cell development and selection associated with lack of apoptosis of thymic cells (lack of tingible body macrophages). Thymic hyperplasia in juvenile animals should be considered among the differential diagnoses of mediastinal masses as a rare cause of respiratory distress in newborn calves.
A 12-year-old, mixed-breed domestic cat was diagnosed with a multicystic hepatic mass via ultrasonographic examination and computer tomography scan. The tumor associated with the left medial liver lobe, and connected by a thin stalk to the hilar region, was surgically removed. The mass was firm, encapsulated, mottled white to red black, multinodular, and cystic. Histologic diagnosis was carcinosarcoma supported by positive immunohistochemistry for cytokeratins and vimentin of atypical neoplastic cell populations. On the basis of morphology, the origin was considered to be in the biliary tract. Biliary carcinosarcoma is a rare neoplasm that occurs in people. The epidemiology and risk factors have not yet been determined, and the prognosis is poor except for cases in which curative resection is performed.
Osteochondromatosis is a condition in which multiple benign, cartilage-capped tumors arise from the surface of bones formed by endochondral ossification. The current report describes the presence of 4 prominent exophytic masses, measuring between 4 and 13 cm in diameter, arising from the surface of the ribs, and located within the thoracic cavity, in a 2-year-old female domestic pig (Sus scrofa domesticus). Histological studies revealed that masses were well-differentiated, cartilage-capped proliferations with an orderly pattern of endochondral mineralization toward deeper areas. The observed gross and microscopic findings are characteristic of osteochondromatosis.
Crotalaria retusa L. (rattleweed), estimated to contain about 4.96% monocrotaline (MCT) in the seed, was associated with a natural poisoning outbreak in goats. The poisoning was experimentally reproduced by the administration of C. retusa seeds containing approximately 4.49% of MCT. Thus, 1 of 3 goats given a single dose of 5 g/kg bodyweight (bw) of seeds (248 mg MCT/kg bw) and 2 goats given a single dose of 347 mg MCT/kg bw showed acute clinical signs and were euthanized 10–11 days after dosing. Clinical signs and gross and histologic lesions were characteristic of acute centrilobular liver necrosis.