Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.     

TB diagnosis in non-human primates: comparison of two interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection

To examine the effect of recombinant bovine interferon-gamma (rbIFN-gamma) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-gamma. All cattle were febrile after inoculation with IFN-gamma and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased for 2-3 days and then their numbers were recovered. The number of gammadelta T cells increased after the fever. In contrast, the number of IgM+ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-gamma administration. These results suggest that IFN-gamma induces the up-regulation of gammadelta T cells, decreases the number of IgM+ lymphocytes and suppresses the growth of BLV in BLV-infected cattle in vivo.     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Comparison of anthelmintic activity of pyrantel, praziquantel, and nitazoxanide against Anoplocephala perfoliata in Horses

Three anthelmintics were compared for efficacy in reducing the egg production of Anoplocephala perfoliata in a herd of central Texas horses. Two trials were run, 1 in mares and the other in weanlings that were diagnosed as being infected with Anoplocephala by recovery of eggs in 5 g of feces with sugar centrifugation. Each animal was evaluated twice before treatment and again twice following treatment (at weeks 2 and 4 after treatment). The criteria for infection were the recovery of eggs on at least 1 occasion before treatment and the finding of eggs on 1 day following treatment. The mares were treated 1 time with either pyrantel pamoate at 13.2 mg/kg, nitazoxanide at 100 mg/kg, praziquantel at 1.23 mg/kg or remained as untreated controls. The weanlings were treated with pyrantel at 13.7 mg/kg nitazoxanide at 100 mg/kg or remained as untreated controls. The percentage reduction of patient infection in mares after treatment with pyrantel was 83%, with nitazoxanide was 78%, and with praziquantel was 83% and in controls was 17%. There was a 75% reduction of patient weanlings treated with pyrantel or nitazoxanide and a 17% reduction in untreated controls. The reduction of infection in all horses treated with any drug was significantly different from controls. All of the drugs were somewhat effective in the control of Anoplocephala, and there were no differences among the drugs in their effectiveness.

Introduction

Anoplocephala perfoliata, the lappeted tapeworm, is an inhabitant of the intestine of equids. Adult tapeworms attach to the intestinal mucosa at the ileocaecal valve and, when present in large numbers, cause edema and hypertrophy of the ileum. The disease manifest by this infection may be inapparent or may give rise to colic (abdominal pain) in the horse apparently from mechanical obstruction or intussusception of the small intestine into the caecocolon.^{1, 2, 3, 4, 5, 6, 7 and 8} The prevalence of infection is geographically variable^{9, 10, 11, 12 and 13} but appears to be increasing,¹⁴ with a much higher rate of infection found with necropsy as opposed to fecal observations. Horses become infected by the ingestion of infected orbatid mites in pastures. Orbatid mites, the intermediate hosts, are predatory and are found in decaying organic material, such as leaf litter. Horses of all ages are infected, but there are lower numbers of clinical cases in horses older than 4 years of age.⁴ The intensity of infection is highest in the late summer and autumn.^{8 and 12} Anthelmintics with reported efficacy against A perfoliata include pyrantel pamoate at 13.2 mg/kg,¹⁰ pyrantel tartrate at 2.6 mg/kg for 30 days,¹⁵ pyrantel embonate at 38 mg/kg,¹⁶ and praziquantel at 1 to 2 mg/kg.^{17 and 18} Nitazoxanide has not been evaluated for Anoplocephala but was included in the trial because of its effects against nematodes and tapeworms in humans.¹⁹ Because Anoplocephala infections may cause disease and there is a perception that current anthelmintics may not be as effective as in the past, a study was done to compare anthelmintics to lower the intensity of fecal egg counts in a herd of horses in central Texas.

Materials and methods

Quarter horse mares and weanlings from a single herd were evaluated with 5 g of feces with a sucrose double centrifugation test to determine whether eggs of Anoplocephala were present.²⁰ Feces from each individual horse were evaluated twice, once approximately 2 weeks before treatment and again on the day of treatment. If Anoplocephala eggs were found on either date, the horse was considered to have positive results. Within each group (mares or weanlings), the treatment selection was randomly allocated as the horses were restrained for treatment. Fecal samples were again evaluated at 14 and 28 days after treatment for the presence or absence of eggs on either day.The dose for each individual horse was determined by chest girth weight tape at the time of treatment. The treatments were as follows: pyrantel pamoate (Strongid-T, Pfizer Animal Health, Exton, Pa) at 13.7 mg/kg via nasogastric intubation (12 mares, 8 weanlings), nitazoxanide oral paste (Nitazoxanide, Idexx Laboratories, Westbrook, Me) at 100 mg/kg (9 mares, 8 weanlings), praziquantel (Droncet injectable, Bayer Corp, Shawnee Mission, Kan) at 1.23 mg/kg via nasogastric intubation (6 mares), and untreated controls (6 mares, 6 weanlings). A 1-tailed Fisher exact test was used to compare rates of infection before and after treatment. If a mare or foal did not have positive results before treatment, it was not evaluated in this study.

Results and discussion

No abnormal clinical signs were seen after treatment with any of the products. Treatment was administered to several additional animals with each product, but they were not included in the analysis if they did not have positive results on 1 of the 2 evaluations before treatment, hence, the different numbers of horses in treatment groups.None of the horses in the trial exhibited clinical signs associated with the infection of A perfoliata. However, before the trial, a mare from the infected herd exhibited signs of colic and Anoplocephala eggs were detected in the feces. Examination of the remainder of the herd gave impetus to the study.Mean egg counts before and after treatment are given in the Table.The presence of strongylate and Parascaris eggs in weanlings served as a control of the methodology of evaluation. The difficulty of finding Anoplocephala eggs has been recognized by several authors,^{5, 8, 13, 14 and 21} but the authors also recognize that when there were greater numbers of parasites there was increased egg production. Therefore, finding of eggs with fecal flotation indicated that there were 20 worms or more. However, there appears to be no correlation between the number of worms and egg counts once the detection threshold is reached,²² so the criterion for evaluation was the presence of eggs in the feces before treatment compared with after treatment. Although mean egg counts were not compared, the number of eggs in each infected horse was less after treatment in all groups compared with untreated controls (Table). The method of evaluation used in this study cannot be equated to those of critical^{10 and 16} or control¹⁴ studies in which horses are killed so that all worms are detected. However, the use of clinical studies to compare compounds is useful in detecting which anthelmintics are likely to be of value against geographically distinct populations of worms. Admittedly, more sampling may have increased the number of horses with positive results, both before and after treatment.     

Phase II Clinical Trial of Carboplatin in Canine Transitional Cell Carcinoma of the Urinary Bladder

Fourteen dogs with histologically-confirmed transitional cell carcinoma (TCC) of the urinary bladder were treated with 300 mg/m² carboplatin every 3 weeks. Response to therapy was assessed with abdominal radiography, double contrast cystography, urinary bladder ultrasonography and thoracic radiography before therapy and at 6–week intervals during therapy. Dogs were monitored for hematologic toxicity with a CBC and platelet count performed immediately before and 10 to 14 days after carboplatin treatment. Tumor responses included progressive disease in 11 dogs and stable disease in 1 dog. Two dogs were euthanized due to carboplatin toxicity before assessment of tumor response. Toxicity included thrombocytopenia with or without neutropenia in 7 dogs and gastrointestinal toxicity in 6 dogs. Carboplatin therapy was not beneficial in the treatment of TCC in the 14 dogs in this study.     

Occurrence and characterization of livestock-associated methicillin-resistant Staphylococcus aureus in pig industries of northern Thailand

This study was conducted to determine the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs, farm workers, and the environment in northern Thailand, and to assess LA-MRSA isolate phenotypic characteristics. One hundred and four pig farms were randomly selected from the 21,152 in Chiang Mai and Lamphun provinces in 2012. Nasal and skin swab samples were collected from pigs and farm workers. Environmental swabs (pig stable floor, faucet, and feeder) were also collected. MRSA was identified by conventional bacterial culture technique, with results confirmed by multiplex PCR and multi locus sequence typing (MLST). Herd prevalence of MRSA was 9.61% (10 of 104 farms). Among pigs, workers, and farm environments, prevalence was 0.68% (two of 292 samples), 2.53% (seven of 276 samples), and 1.28% (four of 312 samples), respectively. Thirteen MRSA isolates (seven from workers, four from environmental samples, and two from pigs) were identified as Staphylococcal chromosomal cassette mec IV sequences type 9. Antimicrobial sensitivity tests found 100% of the MRSA isolates resistant to clindamycin, oxytetracycline, and tetracycline, while 100% were susceptible to cloxacillin and vancomycin. All possessed a multidrug-resistant phenotype. This is the first evidence of an LA-MRSA interrelationship among pigs, workers, and the farm environment in Thailand.     

Prevalence and risk factors for contamination with Listeria monocytogenes of imported and exported meat and fish products in Switzerland, 1992-2000

A total of 2053 import and 164 export samples from 425 production plants were examined over a 9-year period (1992-2000) for the presence of Listeria monocytogenes (L. monocytogenes) in Switzerland. Overall, 282 samples (12.2%) and 85 plants (20.5%) harbored the pathogen. The highest isolation risk was for marinated fish (38%); the lowest was in cured- and dried-meat products.Unconditional fixed-effect logistic regression was used to identify the main hazards associated with the presence of L. monocytogenes. The plant-level model considered potential risk factors for a positive culture operating at the production-plant level by including a random effect of plant and year.Food category was the only significant factor; sampling site, country of origin and season were not significant. Marinated fish was a strong predictor for positive culture, whereas cooked- and cured-meat products were protective. Plant and year effects were significant. Control measures should be focused on specific food items in each production plant.