Who's behind that mask and cape? The Asian leopard cat's Agouti (ASIP) allele likely affects coat colour phenotype in the Bengal cat breed.

Coat colours and patterns are highly variable in cats and are determined mainly by several genes with Mendelian inheritance. A 2-bp deletion in agouti signalling protein (ASIP) is associated with melanism in domestic cats. Bengal cats are hybrids between domestic cats and Asian leopard cats (Prionailurus bengalensis), and the charcoal coat colouration/pattern in Bengals presents as a possible incomplete melanism. The complete coding region of ASIP was directly sequenced in Asian leopard, domestic and Bengal cats. Twenty-seven variants were identified between domestic and leopard cats and were investigated in Bengals and Savannahs, a hybrid with servals (Leptailurus serval). The leopard cat ASIP haplotype was distinguished from domestic cat by four synonymous and four non-synonymous exonic SNPs, as well as 19 intronic variants, including a 42-bp deletion in intron 4. Fifty-six of 64 reported charcoal cats were compound heterozygotes at ASIP, with leopard cat agouti (A(P) (be) ) and domestic cat non-agouti (a) haplotypes. Twenty-four Bengals had an additional unique haplotype (A2) for exon 2 that was not identified in leopard cats, servals or jungle cats (Felis chaus). The compound heterozygote state suggests the leopard cat allele, in combination with the recessive non-agouti allele, influences Bengal markings, producing a darker, yet not completely melanistic coat. This is the first validation of a leopard cat allele segregating in the Bengal breed and likely affecting their overall pelage phenotype. Genetic testing services need to be aware of the possible segregation of wild felid alleles in all assays performed on hybrid cats.

Prevalence and risk factors for common respiratory pathogens within a cohort of pet cats in the UK.

OBJECTIVES: Feline herpesvirus (FHV), feline calicivirus (FCV) and Chlamydia felis are common causes of upper respiratory tract disease (URTD) in cats. Their prevalence in the UK pet cat population has not been reported and little is known regarding the risk factors for their oral carriage. METHODS: Total nucleic acid was extracted from owner-collected buccal swabs (n=600) from cats enrolled in a self-selected longitudinal cohort study. Duplex quantitative PCRs for the detection of FHV and C. felis genomic DNA and reverse-transcriptase quantitative PCRs for the detection of FCV genomic RNA were performed. Duplicates, swabs with insufficient host DNA/RNA, and cats with missing data were excluded. Selected epidemiological data were interrogated using univariable and multi-variable logistic regression modelling to identify risk factors. RESULTS: Data from 430 cats were included in the final statistical model. Of these, 2.1% (n=9/430; 95% CI 1.0% to 3.9%) were positive for FHV, 13.3% (n=57/430; 95% CI 10.2% to 16.8%) positive for FCV and 1.2% (n=5/430; 95% CI 0.4% to 2.7%) positive for C. felis. FCV co-infection was present in five (44%) FHV-positive cats and three (60%) C. felis-positive cats. FCV carriage was more frequent in purebred cats (odds ratio 2.48; 95% CI 1.37 to 4.49) and in cats with current or historical clinical signs compatible with URTD (odds ratio 2.98; 95% CI 1.22 to 7.27). CLINICAL SIGNIFICANCE: FCV was the most frequently encountered URTD pathogen in this sample of cats; this should be noted for disinfectant choice. In cats suspected of having FHV or C. felis infection, assessment for co-infection with FCV is recommended.

First report on the phylogeny of bovine norovirus in Turkey.

Bovine norovirus (BoNoV) is an important cause of diarrhea in calves and has been reported in several countries. The aims of this study were to investigate for the first time the presence of norovirus in Turkish calves by real-time reverse transcription-polymerase chain reaction (qRT-PCR) and to determine the phylogeny of any circulating strains. Fecal samples from 70 diarrheic calves were collected and analysed by SYBR Green qRT-PCR. BoNoV was detected in fecal samples from six calves. The capsid gene was partially sequenced, and phylogenetic analysis was performed. This showed that the six Turkish BoNoVs clustered with the GIII-2 prototype.

Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus.

Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides salivary glands, as well as several members of the D7 family and protease inhibitors with putative anticoagulant activity. In addition, several families of abundant proteins with unknown function were identified including some of the major candidate allergens that cause insect bite hypersensitivity in horses.

Comparison of the value of measurement of serum galactomannan and Aspergillus-specific antibodies in the diagnosis of canine sino-nasal aspergillosis.

Serology is currently used for the diagnosis of canine sino-nasal aspergillosis (SNA). However, the accuracy of serological testing using commercially available, standardized purified antigen preparations of Aspergillus (CAPurAspAg) has only been poorly documented. The aim of the present study was to assess the diagnostic value of an agar-gel double immunodiffusion (AGDD) test and an anti-Aspergillus IgG ELISA, using CAPurAspAg and the commercially available Platelia test for the detection of serum galactomannan. Sera from 17 dogs with SNA, 18 dogs with a nasal tumour (NT), 11 dogs with lymphoplasmacytic rhinitis (LPR) and 33 control dogs were tested with the 3 methods. AGDD result was positive in 76.5% of dogs with SNA, whereas all sera from dogs with non-fungal nasal disease and control dogs were negative. A positive IgG ELISA result was obtained in 88% of dogs with SNA and in 18% of dogs with LPR. All patients with NT and control dogs had a negative IgG ELISA result. The Platelia test was positive in 24% of dogs with SNA, 11% of dogs with NT, 9% of dogs with LPR and 24% of control dogs. The results of this study suggest that (1) the detection of serum Aspergillus-specific antibodies with AGDD or ELISA, using CAPurAspAg, provides excellent specificity and good sensitivity, (2) the specificity is higher for AGDD (100%) than for ELISA (96.8%) while sensitivity is higher for ELISA (88.2%) than for AGDD (76.5%) and (3) serum galactomannan quantification with the Plateliat test is unreliable for the diagnosis of canine SNA.

Antigen receptor V-segment usage in mucosal T cells.

In the accepted model of lymphocyte intestinal homing, naïve T cells recirculate via organized lymphoid tissues, whilst induced effector/memory cells home to the intestinal mucosa. In order to assess the T-cell-receptor repertoire in the intestine and gut-associated lymphoid tissue (GALT), spectratyping was performed on the proximal and the distal intestine, spleen and mesenteric lymph node tissue from six PVG rats. The products were analysed with an automated sequencer and statistical analyses were performed with hierarchical cluster analysis. This demonstrated the presence of a restricted T-cell repertoire in the small intestine compared with that in the mesenteric lymph nodes and the spleen. It also demonstrated marked differences in repertoire between individual, fully inbred rats maintained under apparently identical conditions in the same cage and fed identical diets. In addition, this work demonstrated marked differences between repertoires in the proximal and the distal intestine. Such marked differences are likely to reflect the end result of increasing divergence over time produced by relatively subtle effects of environment and antigenic load. Equally, marked differences in repertoire between small intestinal segments within individual rats indicate selective recruitment or retention of specific clones, presumably antigen-driven.

Is feline foamy virus really apathogenic?

Feline foamy virus (FFV) is a retrovirus commonly found in cats. It is generally thought to be apathogenic, making it a suitable candidate as a gene therapy vector. However, there have been reports of association of FFV with chronic progressive arthritis and a cofactor effect with feline immunodeficiency virus. This study investigated experimental FFV infection and whether this was associated with signs of disease. Eight young specific pathogen free cats were inoculated intramuscularly with FFV. The cats were examined twice weekly and blood and pharyngeal samples were taken. Haematology, biochemistry and FFV quantitative polymerase chain reaction (qPCR) were performed. Tissue samples were also collected throughout the six month period. FFV was initially detected by qPCR in the blood within the first two weeks of infection and viraemia persisted throughout the study. Two peaks of viraemia were observed, at day 20 (80-170FFU/ml blood) and day 155 (332-415FFU/ml blood). FFV was also consistently detected in oropharyngeal samples after day 36. Anti-FFV IgG was detected in all cats by ELISA; antibody levels had an early peak around day 35 and then increased again following the second rise in circulating viral load. All cats remained clinically normal, except for one cat with an unrelated gingivitis. None of the cats developed pyrexia. The biochemical profile and blood cell counts remained within normal limits except for one cat with a persistent eosinophilia. Initial fluctuations in white cell counts settled within three weeks and did not deviate outside of the normal ranges. All tissue samples contained FFV DNA; lymphoreticular tissues, salivary gland and lung had the highest viral loads. Although there were no gross pathological lesions on post mortem examination, histologically a mild glomerulonephritis and a moderate interstitial pneumonia were observed in all cats. We conclude that during the six month period of infection, although cats appeared clinically normal, histopathological changes were observed in the lungs and kidneys. Further investigation of the significance of these changes is warranted before FFV is developed as a vector for gene delivery.

Quantitative real-time RT-PCR measurement of cytokine mRNA expression in the skin of normal cats and cats with allergic skin disease.

Feline allergic skin disease is thought to be associated with dermal infiltration of Th(2) lymphocytes and synthesis of associated cytokines. In this study, real-time RT-PCR assays were developed to measure feline interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the skin of healthy control cats, and in the lesional and non-lesional skin of cats with allergic skin disease. Total RNA was extracted from skin biopsies using the RNeasy Mini Kit with on-column and in-solution DNase digestion steps. cDNA was synthesised using Improm-II reverse transcriptase and random hexamers. Real-time PCR was carried out using an iCycler IQ system (Bio-Rad), and gene-specific primers were designed to span an exon/exon junction of each cytokine gene. Taq-man probes were used to add specificity to the system. Messenger RNA from the housekeeping gene GAPDH was used for normalisation of the cytokine threshold cycle. The eleven cytokine mRNA transcripts quantified were present at varying levels, but there was no apparent difference in expression between normal, non-lesional and lesional skin. TGF-beta represented the most abundant transcript while IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 and TNF-alpha were present at levels approximately 1000-fold less. IL-2 and INF-gamma represented the least abundant templates with no detectable copies in most RNA samples. This quantitative analysis of cytokine mRNA expression in feline skin biopsies has suggested that there is not a simple Th(2) bias in lesional skin of cats with allergic dermatopathies.

Efficacy of pradofloxacin in cats with feline upper respiratory tract disease due to Chlamydophila felis or Mycoplasma infections.

BACKGROUND: Upper respiratory tract disease (URTD) of cats is caused by a number of pathogens, including Chlamydophila felis and Mycoplasma spp. For effective treatment of both infections, doxycycline and enrofloxacin are recommended, but adverse effects limit their use in cats. HYPOTHESIS: That the fluoroquinolone pradofloxacin is effective against C. felis and Mycoplasma infection in cats with URTD or conjunctivitis. ANIMALS: Thirty-nine cats with signs of URTD or conjunctivitis. METHODS: Placebo-controlled, double-blind clinical trial. Cats were randomly entered into 1 of 2 treatment groups: treated PO with either 5 mg/kg pradofloxacin q24h or 5 mg/kg doxycycline q12h for 42 consecutive days. Changes in health status and clinical scores were evaluated. The presence of C. felis and Mycoplasma spp. was determined by quantitative polymerase chain reaction (PCR) and nested PCR of conjunctival swabs, respectively. RESULTS: At the beginning of the study, C. felis and Mycoplasma spp. were detected in 23 and 20 cats, respectively. Cats of both groups responded rapidly with a marked improvement in clinical signs within the 1st week. During treatment with either drug, C. felis DNA copy number declined quickly. Complete elimination of Mycoplasma spp. was achieved in both groups; however, whereas all cats receiving doxycycline eliminated C. felis, 4 cats treated with pradofloxacin remained PCR-positive. CONCLUSION AND CLINICAL IMPORTANCE: This study demonstrates that both pradofloxacin and doxycycline have good efficacy against C. felis and Mycoplasma spp., resulting in a marked improvement of clinical signs. However, C. felis DNA remained in some cats after treatment with pradofloxacin, suggesting that infection might not have been eliminated.

Distinct tissue cytokine and chemokine mRNA expression in canine sino-nasal aspergillosis and idiopathic lymphoplasmacytic rhinitis.

Idiopathic lymphoplasmacytic rhinitis (LPR) and sino-nasal aspergillosis (SNA) are among the most common causes of nasal discharge in dogs. The pathogenesis of both diseases is poorly understood. Some have proposed that LPR is a chronic inflammatory response to an inhaled irritant, pollutant or allergen, but others suggest that most cases of LPR constitute undiagnosed cases of SNA. Local immune dysfunction is thought to permit opportunist infection in canine SNA. This study investigates the nature of the local tissue immune response mounted in canine LPR and SNA in order to determine whether these diseases have similar or distinct pathogenesis. Quantitative reverse transcriptase polymerase chain reaction was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify messenger RNA (mRNA), encoding a panel of cytokines and chemokines. SNA was associated with significantly increased expression of mRNA encoding interleukin (IL)-6, IL-8, IL-10, IL-12p19, IL-12p35, IL-12p40, IL-18, IFN-gamma, TNF-alpha, TGF-beta, eotaxin-2 and all four monocyte chemoattractant proteins (MCPs) relative to controls. LPR was associated with significantly increased expression of mRNA encoding IL-5, IL-8, IL-10, IL-12p19, IL-12p40, IL-18, TNF-alpha, TGF-beta, MCP-2 and MCP-3 relative to controls. There was significantly more expression of mRNA encoding IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-18, IFN-gamma, TNF-alpha, TGF-beta and all MCPs, and significantly less expression of IL-5 in dogs with SNA than in dogs with LPR. Thus, the profile of cytokine and chemokine gene expression in the nasal mucosa is different in dogs with LPR when compared to dogs with SNA. A partial Th2 immune response appears to be mounted in the nasal mucosa of dogs with LPR, whereas the mucosal immune response in canine SNA is of the Th1 type. Increase in IL-10 and TGF-beta transcripts in dogs with SNA is thought to be implicated in the failure to clear the Aspergillus infection. These results constitute the first evidence that the pathogenesis of canine LPR and SNA is distinct.

Measurement of cytokine mRNA expression in intestinal biopsies of cats with inflammatory enteropathy using quantitative real-time RT-PCR.

Inflammatory bowel disease (IBD) is a common condition in cats characterised by infiltration of inflammatory cells into the intestinal mucosa. In this study, real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantify cytokine messenger RNA (mRNA) expression in intestinal biopsies from cats. Biopsies were collected from seven cats with chronic diarrhoea and histologically confirmed IBD, five cats with chronic diarrhoea due to non-IBD gastrointestinal (GI) disease, and nine clinically normal cats with or without subclinical inflammatory changes in small intestine. Real-time RT-PCR was developed for quantification of mRNA encoding interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a 'housekeeper' gene. All real-time PCR efficiencies were>90% (range 90.4-102%) with correlation coefficients >0.99 (range 0.998-1). The results of the study were analyzed on the basis of either clinical presentation or histopathological evidence of intestinal inflammation. The former analysis showed that mRNA encoding IL-10 and TGF-beta (immunoregulatory cytokines), and IL-6, IL-18, TNF-alpha and IL-12 p40 (Th1 and pro-inflammatory cytokines) was significantly higher in clinically normal cats and cats with IBD when compared to cats with other GI diseases. IL-5 mRNA was significantly higher in cats with IBD compared to clinically normal cats. IL-2 mRNA was significantly lower in cats with non-IBD GI disease than in clinically normal cats. Analysis on the basis of histopathological change revealed that cats with intestinal inflammation had significantly more transcription of genes encoding IL-6, IL-10, IL-12p40, TNF-alpha and TGF-beta than those with normal intestinal morphology. The results suggest that immune dysregulation plays a role in feline IBD and that IBD in cats has a complicated pathogenesis with both pro-inflammatory and immunoregulatory features.

Analysis of risk factors and prevalence of haemoplasma infection in dogs.

Mycoplasma haemocanis (Mhc) and 'Candidatus Mycoplasma haematoparvum' (CMhp) are canine haemoplasma species that can induce anaemia in immunocompromised and/or splenectomised dogs. This study aimed to determine the prevalence and phylogeny of canine haemoplasma species in dogs from Nigeria and describe any risk factors for infection. Canine haemoplasma species-specific and generic haemoplasma qPCR assays were used. The species-specific qPCR assays found Mhc infection in 18 of 245 dogs (7.3%), and CMhp infection in only one dog (0.4%). The generic haemoplasma qPCR assays were positive in 44 of 245 (17.9%) dogs. Twenty-five dogs had discordant qPCR results in that they were generic haemoplasma qPCR positive but species-specific qPCR negative. Further evaluation of these dogs by 16S rDNA sequencing gave limited results but 5 were confirmed to be infected with non-haemoplasma species: 2 Anaplasma phagocytophilum, 1 Anaplasma ovis, 1 Serratia marcescens and 1 Aerococcus spp. The 16S rRNA gene sequences from Mhc species showed>99.8% identity with each other and>99.6% identity with GenBank sequences, and resided in a single clade with other global Mhc and Mycoplasma haemofelis sequences, indicating low 16S rRNA genetic variability amongst this canine haemoplasma species.

Detection of allelic variants of the canine IGHA gene by fluorescence resonance energy transfer melting temperature examination.

The fluorescence resonance energy transfer (FRET) dual hybridisation probe system has been used for the detection of the accumulation of target DNA during real-time PCR and for the identification of nucleotide polymorphisms through examination of melt curves. This system involves the use of two oligonucleotide probes which are located close to each other and are complementary to an internal segment of a target DNA of interest. Four allelic variants of the gene encoding the hinge region of the immunoglobulin A (IgA) heavy chain (IGHA) have been so far identified in the dog and this variability is due to a combination of single nucleotide polymorphisms and insertion/deletion of nucleic acid motifs. An individual dog may be homozygous or heterozygous for these allelic variants. The purpose of this study was to develop a FRET-based dual probe melting temperature assay to identify the alleles present within an individual dog and to use this assay to determine the frequency of the four allelic variants in different breeds within the canine population. A single pair of oligonucleotide probes were designed that were able to discriminate between the four allelic variants in both homozygous and heterozygous individuals. The genotype of 96 DNA samples obtained from various purebreeds of dogs was determined using this FRET assay. The frequency of each allele differed between the breed groups. The results of this study indicate that it is possible to distinguish relatively complex gene polymorphisms using a single set of oligonucleotide probes. Furthermore, any future comparison of IGHA genotypes between normal and diseased dog populations must take into account the breed variation in allelic frequency.

Measurement of serum immunoglobulin E (IgE) specific for house dust mite antigens in normal cats and cats with allergic skin disease.

The purpose of this study was to determine whether cats with allergic skin disease have significant concentrations of serum Immunoglobulin E (IgE) specific for antigens derived from the house dust mites (HDM) Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP). Enzyme-linked immunosorbent assays (ELISA) were developed for this purpose. Binding of serum allergen-specific IgE was detected via the use of biotinylated Fc-epsilon receptor alpha chain protein (FcvarepsilonRIalpha). Following optimisation of the assay, serum samples from 59 cats with allergic skin disease and 54 clinically normal cats were screened. Results were expressed as ELISA units per ml (EU/ml) compared to a standard curve. Serological findings were correlated with the clinical presentation of affected cats. Cats with symptoms of feline allergic skin disease were grouped as follows: self-induced alopecia without lesions (group 1), papulocrusting dermatitis (group 2), eosinophilic granuloma complex (group 3), papular/ulcerative dermatitis of head and neck/facial dermatitis (group 4), and a combination of symptoms (group 5). Control normal cats comprised the final group (group 6). The Kruskal-Wallis test was used for statistical analysis. There was no significant difference between groups for DF- and DP-specific IgE concentrations with a p-value of 0.875 and 0.705, respectively. Although the FcvarepsilonRIalpha-based ELISA was able to detect house dust mite-specific feline IgE, the presence of this allergen-specific IgE correlates poorly with the presence of clinical manifestations of allergic skin disease. The results of this study question the clinical relevance of house dust mite-specific IgE in feline allergic skin disease.

Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

Brain tissue fragments in jugular vein blood of cattle stunned by use of penetrating or nonpenetrating captive bolt guns.

Although the incidence of bovine spongiform encephalopathy in cattle continues to decline in the United Kingdom, it remains important to maintain vigilance of all potential routes of transmission of infection to humans. Initial studies have demonstrated a potential risk of carcass contamination with brain tissue following the use of captive bolt gun stunning in cattle. The objective of this study was to further explore these initial findings particularly in regard to captive bolt guns currently in use in the United Kingdom. Brain tissue fragments or elevated levels of a marker protein for brain tissue were detected in venous blood samples from 4% (95% confidence interval, 1.6 to 9.8%) of cattle stunned by penetrating captive bolt gun and from 2% (95% confidence interval, 0.6 to 7%) of those stunned by nonpenetrating captive bolt gun.

Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis and Bordetella bronchiseptica in cats: experience from 218 European catteries.

A full history of the management practices and the prevalence of upper respiratory tract disease (URTD) at 218 rescue shelters, breeding establishments and private households with five or more cats was recorded. Oropharyngeal and conjunctival swabs and blood samples were taken from 1748 cats. The prevalences of feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydophila felis and Bordetella bronchiseptica were determined by PCR on swab samples. An ELISA was applied to determine the prevalence of antibodies to B. bronchiseptica. The rates of detection by PCR of each pathogen in the cats in catteries with and without ongoing URTD were, respectively, FHV 16 per cent and 8 per cent; FCV 47 per cent and 29 per cent; C. felis 10 per cent and 3 per cent; and B. bronchiseptica 5 per cent and 1.3 per cent; the seroprevalences of B. bronchiseptica were 61 per cent and 41 per cent, respectively. There was evidence that FHV, FCV and B. bronchiseptica played a role in URTD. The risk factors associated with the disease were less than excellent hygiene, contact with dogs with URTD, and larger numbers of cats in the cattery or household.

A retrospective clinical and epidemiological study on feline coronavirus (FCoV) in cats in Istanbul, Turkey.

The presence of antibodies to feline coronavirus (FCoV) and feline immunodeficiency virus (FIV), together with feline leukemia virus (FeLV) antigen was investigated in 169 ill household and stray cats attending a veterinary surgery in Istanbul in 2009-14. The estimated FCoV and FIV seroprevalence (95% confidence intervals) were 37% (30-45%) and 11% (6-16%), respectively and FeLV prevalence was 1% (0-3%). FCoV seroprevalence increased until 2 years of age, was highest in 2014 and among household cats living with other cats and with outdoor access, and was lower in FIV seropositive compared to seronegative cats. Symptoms typically associated with wet feline infectious peritonitis (FIP) including ascites, abdominal distention or pleural effusion, coupled in many cases with non-antibiotic responsive fever, were observed in 19% (32/169) of cats, and 75% (24/32) of these cats were FCoV seropositive. FCoV seropositivity was also associated with a high white blood cell count, high plasma globulin, low plasma albumin and low blood urea nitrogen. The percentage of FCoV seropositive and seronegative cats that died in spite of supportive veterinary treatment was 33% (21/63) and 12% (13/106), respectively. These results indicate that FCoV is widespread and has a severe clinical impact in cats from Istanbul. Moreover, the incidence of FCoV infections could be rising, and in the absence of effective vaccination cat owners need to be made aware of ways to minimize the spread of this virus.

Real-time RT-PCR: considerations for efficient and sensitive assay design.

Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taq-man probes with a deoxyguanosine base at the 5' end fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer-dimer formation associated with RT enzymes is described.

Methods for detection of haematogenous dissemination of brain tissue after stunning of cattle with captive bolt guns.

Because of concern that the stunning of cattle with captive bolt guns (CBGs) could, if used on an animal with bovine spongiform encephalopathy (BSE), cause embolism of infective brain tissue and carcass contamination, the Ministry of Agriculture, Food and Fisheries commissioned research to assess the risk of haematogenous dissemination of CNS material after stunning. We have devised two methods to investigate this risk. The first involves the concentration of embolic tissue in buffy coat Cytoblocks that can be embedded for sectioning, microscopy and immunocytochemistry. The second method is an ELISA for the presynaptic protein, syntaxin 1B. The methods were validated by analysis of several bovine tissues, including blood samples deliberately contaminated with brain. We then studied jugular venous blood obtained before and after the stunning of 60 cattle with CBGs. Samples obtained, after stunning, from five of the cattle contained CNS tissue within the Cytoblocks and yielded positive syntaxin assays. Syntaxin was also detected in samples from one other animal that had been stunned with a pneumatically operated CBG. The described methods should allow an assessment of the risk of neuroembolism associated with different types of CBG and may also be useful in other contexts.