Euthanization methods influence cytokine mRNA expression levels in age 0 year Oncorhynchus mykiss.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Effects of oral tetrachlorvinphos fly control (Equitrol) administration in horses: physiological and behavioural findings.

Highly reactive horses may pose risks to humans involved in equestrian activities. Among the factors that may affect horses' reactivity to external stimuli are pesticides used for fly control in equine facilities. The organophosphorus (OP) insecticide tetrachlorvinphos (TCVP) is used as a feed-through larvicide to prevent completion of the fly larval life cycle in horse manure. TCVP exerts its effect by inhibiting the enzyme cholinesterase (ChE) leading to the accumulation of the neurotransmitter acetylcholine (AChE) in synapses of the central and peripheral nervous systems. The aim of the present study was to investigate alterations of whole-blood ChE levels associated with feeding a commercially available product (Equitrol, Farnam Companies, Inc.) to horses for fly control. A second aim was to report neurological, physiological and behavioural findings in addition to profiles of selected immune markers (IFN-gamma, IL-12p40 and COX-2) and serum thyroid hormones during and after a 30-day treatment period of TCVP feeding. The results indicated significant decreases in whole-blood ChE activity and concomitant behavioural alterations, manifested as increased reactivity and decreased controllability in treated horses. No changes were detected in physiological or neurological parameters, immune markers or thyroid hormones in treated (n=6) or control (n=4) horses during the course of the study.

Enteric coronavirus infection in adult horses.

A new enteric virus of adult horses, equine coronavirus (ECoV), has recently been recognized. It is associated with fever, lethargy, anorexia, and less frequently, colic and diarrhea. This enteric virus is transmitted via the feco-oral route and horses become infected by ingesting fecally contaminated feed and water. Various outbreaks have been reported since 2010 from Japan, Europe and the USA. While the clinical signs are fairly non-specific, lymphopenia and neutropenia are often seen. Specific diagnosis is made by the detection of ECoV in feces by either quantitative real-time PCR, electron microscopy or antigen-capture ELISA. Supportive treatment is usually required, as most infections are self-limiting. However, rare complications, such as endotoxemia, septicemia and hyperammonemia-associated encephalopathy, have been reported, and have been related to the loss of barrier function at the intestinal mucosa. This review article will focus on the latest information pertaining to the virus, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

Use of quantitative real-time PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles.

BACKGROUND: In recent years, molecular approaches have been able to characterise the viability of equine upper respiratory tract pathogens using absolute molecular quantitation as well as detection of transcripts for virulence genes. OBJECTIVES: The objective of this study was to investigate molecular surrogates for S. equi subspecies equi (S. equi) viability in biological samples from horses with strangles. STUDY DESIGN: Retrospective cross-sectional study. METHODS: S. equi culture-positive and culture-negative upper airway secretions were assessed by qPCR at the genomic (gDNA) and complimentary DNA (cDNA) level for various target genes (SeM, SEQ2190, eqbE and szpSe). Absolute quantitation was performed using standard curves, and the results were expressed as number of S. equi target genes per µl of gDNA or cDNA. Additionally, the presence or absence of S. equi gene expression for the various target genes was assessed and compared with the culture results. RESULTS: While all 21 culture-positive samples tested S. equiqPCR positive, up to 43.7 and 18.9% of 64 culture-negative samples tested qPCR positive at the gDNA and cDNA level, respectively. Significant differences in absolute quantitation for S. equi at the gDNA level were found between culture-positive and culture-negative samples. When absolute quantitation of S. equi target genes at the gDNA level was assessed with the presence or absence of transcripts, there was a significantly higher S. equi target gene number in samples with expression of transcripts compared with samples with no expression of transcripts. MAIN LIMITATIONS: The lack of standardisation of samples collected in the field and the delay from sample collection to samples processing may have negatively affected the cultivability of S. equi and mRNA quality. CONCLUSIONS: Molecular viability for S. equi can be investigated by determining absolute quantitation and/or by detecting mRNA for specific target genes. However, veterinarians have to be cautioned that any qPCR-positive result for S. equi needs to be taken seriously and trigger biosecurity protocols aimed at reducing spread.

Prevalence of enteropathogens in cats with and without diarrhea in four different management models for unowned cats in the southeast United States.

The objective of this study was to determine the prevalence of enteropathogens in cats with and without diarrhea in four different models for managing unowned cats: short-term animal shelter, long-term sanctuary, home-based foster care, and trap-neuter-return. Fecal samples from 482 cats, approximately half of the cats with normal fecal consistency and half with diarrhea, were tested by zinc sulfate centrifugation and by real-time PCR for a panel of enteropathogens. At least one enteropathogen of feline or zoonotic importance was detected in a majority of cats, regardless of management model. For most enteropathogens, the presence or absence of diarrhea was not significantly associated with infection, the exceptions being Tritrichomonas foetus in sanctuary cats with diarrhea (26%) and normal fecal consistency (10%), respectively (P≤0.04), and feline coronavirus in foster cats (80% and 58%) (P≤0.001). The types of enteropathogens detected were related to the type of management model, e.g., viral and protozoal infections were most common in shelters, sanctuaries, and foster homes (confinement systems), whereas helminth infections were most common in trap-neuter-return programs (free-roaming cats). These results suggest that management practices for unowned cats are inadequate for control of enteropathogens and that the presence of diarrhea is a poor indicator of enteropathogen carriage. Risk-management strategies to reduce transmission to people and other animals should focus on sanitation, housing, compliance with preventive care guidelines, periodic surveillance, response to specific enteropathogens, humane population management of free-roaming community cats, public health education, and minimizing the duration and number of cats in mass confinement.

Molecular quantitative analysis of human viruses in California stormwater.

Many human pathogenic viruses are transmitted via the oral-fecal route and water is one possible vector, representing a risk for public health. Sixty-one large-volume water samples from storm drains in California were processed by a two-step hollow fiber ultrafiltration procedure followed by molecular analysis for human enterovirus and adenovirus types. Each sample was spiked with a surrogate, the benign bacteriophage PP7. Both surrogate and human viruses were quantified by newly designed TaqMan PCR assays. Equations were developed that account for the main variables in the procedure: recovery of the ultrafiltration, efficiency of nucleic acid extraction, and effect of inhibitors on the amplification of viral targets. Adenovirus 40/41 was detected in one sample at 230 genomes per liter, and no other adenovirus or enterovirus types were found. Samples that resulted in nondetects are reported together with the corresponding sample-specific limit of detection (S(LOD)), a useful tool when estimating the public health risk associated with the contact or ingestion of water. Virus concentrations did not correlate with traditional viable indicator concentrations or any of the physicochemical parameters measured. In contrast, coliform concentrations were correlated with total suspended solids. To our knowledge, this is the first study where all factors known to influence limits of detection have been investigated and integrated into equations that are widely applicable to the quantification of viruses or other microbial targets by PCR.

Comparison of five real-time PCR assays for detecting virulence genes in isolates of Escherichia coli from septicaemic neonatal foals.

Fifty-five isolates of Escherichia coli from septicaemic neonatal foals were used to validate five real-time pcr assays targeting different known virulence factor genes: curli fibre (csgD), ferric hydroxamate uptake (fhuA), type 1A pilin (fimA), aerobactin (lutA) and yersiniabactin (fyuA). A pcr assay targeting a universal sequence of the bacterial 16S rrna gene served as quality control. The pcr assays showed good analytical specificity and sensitivity on the basis of sequencing the pcr products, their lack of cross-reactivity with non-E coli organisms, high amplification efficiency and a limit of detection as low as 25 E coli colony-forming units. There were differences between the detection rates and amplification efficiencies for the five virulence genes. The pcr assays targeting genes csgD, fhuA and fyuA were able to detect all 55 E coli isolates, with gene csgD having the best amplification efficiency. The lowest detection rate and amplification efficiency of the E coli isolates was found for the lutA gene.

Role of canine circovirus in dogs with acute haemorrhagic diarrhoea.

Canine circovirus (CanineCV) has been detected in some dogs with severe haemorrhagic diarrhoea, but its pathogenic role is unclear. This study evaluated a suspected association between the presence of CanineCV and acute haemorrhagic diarrhoea syndrome (AHDS) in dogs. The prevalence of CanineCV in dogs with AHDS was compared with that in healthy dogs and those infected with canine parvovirus (CPV). Additionally, time to recovery and mortality rate were compared between CanineCV-positive and CanineCV-negative dogs. Faecal samples of dogs with AHDS (n=55), healthy dogs (n=66) and dogs infected with CPV (n=54) were examined by two real-time TaqMan PCR assays targeting the replicase and capsid genes of CanineCV. CanineCV was detected in faecal samples of two dogs with AHDS, three healthy controls and seven dogs infected with CPV. Among the three groups, there was no significant difference in prevalence of CanineCV. CPV-infected animals that were coinfected with CanineCV had a significantly higher mortality rate compared with those negative for CanineCV. CanineCV does not appear to be the primary causative agent of AHDS in dogs, but might play a role as a negative co-factor in disease outcome in dogs with CPV infection.