Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits.

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

A domestic cat whole exome sequencing resource for trait discovery.

Over 94 million domestic cats are susceptible to cancers and other common and rare diseases. Whole exome sequencing (WES) is a proven strategy to study these disease-causing variants. Presented is a 35.7 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. Whole exome sequencing was conducted on 41 cats with known and unknown genetic diseases and traits, of which ten cats had matching whole genome sequence (WGS) data available, used to validate WES performance. At 80 × mean exome depth of coverage, 96.4% of on-target base coverage had a sequencing depth > 20-fold, while over 98% of single nucleotide variants (SNVs) identified by WGS were also identified by WES. Platform-specific SNVs were restricted to sex chromosomes and a small number of olfactory receptor genes. Within the 41 cats, we identified 31 previously known causal variants and discovered new gene candidate variants, including novel missense variance for polycystic kidney disease and atrichia in the Peterbald cat. These results show the utility of WES to identify novel gene candidate alleles for diseases and traits for the first time in a feline model.

Evaluation of Interferon-Gamma Polymorphisms as a Risk Factor in Feline Infectious Peritonitis Development in Non-Pedigree Cats-A Large Cohort Study.

Feline infectious peritonitis (FIP) is a common infectious cause of death in cats, with heritable host factors associated with altered risk of disease. To assess the role of feline interferon-gamma gene (fIFNG) variants in this risk, the allele frequencies of two single nucleotide polymorphisms (SNPs) (g.401 and g.408) were determined for non-pedigree cats either with confirmed FIP (n = 59) or from the general population (cats enrolled in a large lifetime longitudinal study; n = 264). DNA was extracted from buccal swabs or tissue samples. A pyrosequencing assay to characterize the fIFNG SNPs was designed, optimized and subsequently performed on all samples. Genotype and allele frequency were calculated for each population. Characterization of the target SNPs was possible for 56 of the cats with FIP and 263 of the cats from the general population. The SNPs were in complete linkage disequilibrium with each other. There was an association between FIP status and genotype (χ2; p = 0.028), with a reduced risk of developing FIP (χ2; p = 0.0077) associated with the genotype TT at both positions. These results indicate that, although fIFNG variants may be associated with altered risk of disease, the prevalence of individual variants within both populations limits application of their characterization to breeding purposes.

Evaluation of polymorphisms in inflammatory mediator and cellular adhesion genes as risk factors for feline infectious peritonitis.

OBJECTIVES: Feline infectious peritonitis (FIP) is a high mortality infectious disease. Single nucleotide polymorphisms (SNPs) in the genes encoding interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN; CD209) have been associated with increased and decreased risk of developing FIP. This study was designed to determine whether these associations were present in a UK population of pedigree cats using samples from cats euthanased with a confirmed diagnosis (FIP, n = 22; non-FIP, n = 10) or clinically healthy cats over 11 years of age (n = 3). METHODS: DNA was extracted from tissue (n = 32) or blood (n = 3) and PCR performed for regions of IFNG, TNFA and CD209. PCR amplicons were sequenced, each SNP genotype was determined, and genotype/allele frequency for each SNP and FIP status were compared. RESULTS: No significant association was found between the genotype and FIP status for any SNP analysed. There was a trend for the heterozygous CT genotype at both IFNG g.401 and IFNG g.408 to be associated with FIP (P = 0.13), but this genotype was also found in a substantial proportion of non-FIP cats. There was also a trend for the heterozygous CT genotype at IFNG g.428 to be associated with FIP (P = 0.06), although most cats with FIP had the CC genotype at this locus. No associations were found between any allele at TNFA g.-421, CD209 g.1900, CD209 g.2276, CD209 g.2392 and CD209 g.2713 and FIP. CONCLUSIONS AND RELEVANCE: The use of the IFNG, TNFA and CD209 SNPs described to predict the risk of FIP cannot currently be recommended.

Author Correction: Applications and efficiencies of the first cat 63 K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Author Correction: Applications and efficiencies of the first cat 63K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Applications and efficiencies of the first cat 63K DNA array.

The development of high throughput SNP genotyping technologies has improved the genetic dissection of simple and complex traits in many species including cats. The properties of feline 62,897 SNPs Illumina Infinium iSelect DNA array are described using a dataset of over 2,000 feline samples, the most extensive to date, representing 41 cat breeds, a random bred population, and four wild felid species. Accuracy and efficiency of the array's genotypes and its utility in performing population-based analyses were evaluated. Average marker distance across the array was 37,741 Kb, and across the dataset, only 1% (625) of the markers exhibited poor genotyping and only 0.35% (221) showed Mendelian errors. Marker polymorphism varied across cat breeds and the average minor allele frequency (MAF) of all markers across domestic cats was 0.21. Population structure analysis confirmed a Western to Eastern structural continuum of cat breeds. Genome-wide linkage disequilibrium ranged from 50-1,500 Kb for domestic cats and 750 Kb for European wildcats (Felis silvestris silvestris). Array use in trait association mapping was investigated under different modes of inheritance, selection and population sizes. The efficient array design and cat genotype dataset continues to advance the understanding of cat breeds and will support monogenic health studies across feline breeds and populations.

Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis.

Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

Whole genome sequencing in cats, identifies new models for blindness in AIPL1 and somite segmentation in HES7.

BACKGROUND: The reduced cost and improved efficiency of whole genome sequencing (WGS) is drastically improving the development of cats as biomedical models. Persian cats are models for Leber's congenital amaurosis (LCA), the most severe and earliest onset form of visual impairment in humans. Cats with innocuous breed-defining traits, such as a bobbed tail, can also be models for somite segmentation and vertebral column development. METHODS: The first WGS in cats was conducted on a trio segregating for LCA and the bobbed tail abnormality. Variants were identified using FreeBayes and effects predicted using SnpEff. Variants within a known haplotype block for cat LCA and specific candidate genes for both phenotypes were prioritized by the predicted variant effect on the proteins and concordant segregation within the trio. The efficiency of WGS of a single trio of domestic cats was evaluated. RESULTS: A stop gain was identified at position c.577C > T in cat AIPL1, a predicted p.Arg193*. A c.5A > G variant causing a p.V2A was identified in HES7. The variants segregated concordantly in a Persian - Japanese bobtail pedigree. Over 1700 cats from 40 different breeds and populations were genotyped for the AIPL1 variant, defining an allelic frequency in only Persian -related breeds of 1.15%. A sub-set of cats was genotyped for the HES7 variant, supporting the variant as private to the Japanese bobtail breed. Approximately 18 million SNPs were identified for application in cat research. The cat AIPL1 variant would have been considered a high priority variant for evaluation, regardless of a priori knowledge from previous genetic studies. CONCLUSIONS: This study represents the first effort of the 99 Lives Cat Genome Sequencing Initiative to identify disease--causing variants in the domestic cat using WGS. The current cat reference assembly is efficient for gene and variant identification. However, as the feline variant database improves, development of cats as biomedical models for human disease will be more efficient, providing an alternative, large animal model for drug and gene therapy trials. Undiagnosed human patients with early-onset blindness should be screened for this AIPL1 variant. The HES7 variant should further calibrate the somite segmentation clock.

Aristaless-Like Homeobox protein 1 (ALX1) variant associated with craniofacial structure and frontonasal dysplasia in Burmese cats.

Frontonasal dysplasia (FND) can have severe presentations that are medically and socially debilitating. Several genes are implicated in FND conditions, including Aristaless-Like Homeobox 1 (ALX1), which is associated with FND3. Breeds of cats are selected and bred for extremes in craniofacial morphologies. In particular, a lineage of Burmese cats with severe brachycephyla is extremely popular and is termed Contemporary Burmese. Genetic studies demonstrated that the brachycephyla of the Contemporary Burmese is a simple co-dominant trait, however, the homozygous cats have a severe craniofacial defect that is incompatible with life. The craniofacial defect of the Burmese was genetically analyzed over a 20 year period, using various genetic analysis techniques. Family-based linkage analysis localized the trait to cat chromosome B4. Genome-wide association studies and other genetic analyses of SNP data refined a critical region. Sequence analysis identified a 12bp in frame deletion in ALX1, c.496delCTCTCAGGACTG, which is 100% concordant with the craniofacial defect and not found in cats not related to the Contemporary Burmese.

Genotyping coronaviruses associated with feline infectious peritonitis.

Feline coronavirus (FCoV) infections are endemic among cats worldwide. The majority of infections are asymptomatic or result in only mild enteric disease. However, approximately 5 % of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs: three from faecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of 8 weeks at a single-site cat rescue and rehoming centre in the UK. We found amino acid differences located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminated between the genomes derived from the faecal samples and the genomes derived from the tissue lesion samples. In this study, two amino acid differences fully discriminated the two classes of genomes: these were both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP.

Protective immunity against infection with Mycoplasma haemofelis.

Hemoplasmas are potentially zoonotic mycoplasmal pathogens, which are not consistently cleared by antibiotic therapy. Mycoplasma haemofelis is the most pathogenic feline hemoplasma species. The aim of this study was to determine how cats previously infected with M. haemofelis that had recovered reacted when rechallenged with M. haemofelis and to characterize the immune response following de novo M. haemofelis infection and rechallenge. Five specific-pathogen-free (SPF)-derived naive cats (group A) and five cats that had recovered from M. haemofelis infection (group B) were inoculated subcutaneously with M. haemofelis. Blood M. haemofelis loads were measured by quantitative PCR (qPCR), antibody response to heat shock protein 70 (DnaK) by enzyme-linked immunosorbent assay (ELISA), blood lymphocyte cell subtypes by flow cytometry, and cytokine mRNA levels by quantitative reverse transcriptase PCR. Group A cats all became infected with high bacterial loads and seroconverted, while group B cats were protected from reinfection, thus providing the unique opportunity to study the immunological parameters associated with this protective immune response against M. haemofelis. First, a strong humoral response to DnaK was only observed in group A, demonstrating that an antibody response to DnaK is not important for protective immunity. Second, proinflammatory cytokine interleukin-6 (IL-6) mRNA levels appeared to increase rapidly postinoculation in group B, indicating a possible role in protective immunity. Third, an increase in IL-12p35 and -p40 mRNA and decrease in the Th2/Th1 ratio observed in group A suggest that a Th1-type response is important in primary infection. This is the first study to demonstrate protective immunity against M. haemofelis reinfection, and it provides important information for potential future hemoplasma vaccine design.

Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis.

Recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (FCoV), which results in an amino acid change from methionine to leucine at position 1058, may be associated with feline infectious peritonitis (FIP). Tissue and faecal samples collected post mortem from cats diagnosed with or without FIP were subjected to RNA extraction and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect FCoV RNA. In cats with FIP, 95% of tissue, and 81% of faecal samples were PCR-positive, as opposed to 22% of tissue, and 60% of faecal samples in cats without FIP. Relative FCoV copy numbers were significantly higher in the cats with FIP, both in tissues (P < 0.001) and faeces (P = 0.02). PCR-positive samples underwent pyrosequencing encompassing position 1058 of the FCoV spike protein. This identified a methionine codon at position 1058, consistent with the shedding of an enteric form of FCoV, in 77% of the faecal samples from cats with FIP, and in 100% of the samples from cats without FIP. In contrast, 91% of the tissue samples from cats with FIP and 89% from cats without FIP had a leucine codon at position 1058, consistent with a systemic form of FCoV. These results suggest that the methionine to leucine substitution at position 1058 in the FCoV spike protein is indicative of systemic spread of FCoV from the intestine, rather than a virus with the potential to cause FIP.

Frequency and phylogeny of norovirus in diarrheic children in Istanbul, Turkey.

BACKGROUND: Norovirus (NoV) is recognised as one of the most common causes of foodborne infections. Contaminated shellfish, food, water and hospitals are well documented sources of the virus. OBJECTIVE: NoV in diarrheic children has not previously been investigated in Istanbul, Turkey, hence the aim of this study was to detect and investigate the frequency and phylogeny of human NoV genogroups I and II in children with acute gastroenteritis. STUDY DESIGN: 238 stool samples were collected from diarrheic children from 2 hospitals (Cerrahpasa Medical School and Haseki) in Istanbul and analysed by ELISA, RT-PCR and real-time RT-PCR using both SYBR Green and probe-based assays for human NoV. Primers targeting the RNA-polymerase gene were used for RT-PCR to allow DNA sequencing of Turkish NoV strains and phylogenetic analysis to be performed. RESULTS: NoV GII was detected in 36 (15.1%) of 238 samples by SYBR Green real-time RT-PCR, 10.9% by a probe-based real-time RT-PCR and 10.5% by ELISA (Ridascreen). Genogroup II (GII) the Turkish NoVs clustered with including GII4 (72.2%), GII16 (5.5%), GIIb (16.7%) and GIIe (5.5%). Two variants of GII4 (GII4-2006b and GII4-2008), GII16 and recombinant noroviruses (GIIb and GIIe) were identified. CONCLUSION: This study shows a high frequency and genetic diversity of NoV GII infections in children with acute gastroenteritis in Istanbul, Turkey.

Detection of cold-tolerant clostridia other than Clostridium estertheticum in raw vacuum-packed chill-stored meat.

Samples from raw chill-stored vacuum-packed beef, lamb and venison or the meat processing environment, associated with a spoilage problem, but negative for Clostridium estertheticum using a specific real-time PCR test, were examined for other Clostridium spp. using direct 16S rDNA PCR-RFLP and sequencing. Of 291 samples tested by PCR, presence of clostridia was indicated in 123 and there was sufficient PCR product in 35 to be further investigated. Presence of Clostridium spp. was confirmed by RFLP and sequencing in 25/35 samples (11 of 14 incidents). Species detected in spoiled meat were (incidents): Clostridium tagluense-like (4), Clostridium putrefaciens (2), Clostridium algidicarnis (3), Clostridium frigoris/estertheticum-like (3) and Clostridium. gasigenes (2). More than one species was detected in some incidents. All of the above species have previously been associated with spoiled meat apart from the Cl. tagluense-like species. Clostridia were also confirmed in 4/7 samples from the environment, with two Cl. frigoris/estertheticum-like and two mesophilic species of Clostridium. Our study showed that, cold-tolerant Clostridium species other than Cl. estertheticum are occasionally associated with spoiled vacuum-packed meat, particularly lamb. Further studies are required to confirm the exact identity of the Cl. tagluense-like species and its role in meat spoilage.

Polymerase chain reaction survey of feline haemoplasma infections in Greece.

The aim of this study was to use real-time polymerase chain reaction assays to determine the prevalence of three haemoplasma species in cats from Greece and to evaluate possible associations between haemoplasma infection and age, gender, feline immunodeficiency virus/feline leukaemia virus (FIV/FeLV) status and packed cell volume (PCV). Ninety-seven cats (24 ill anaemic, 55 ill non-anaemic, 18 healthy non-anaemic) were included in the study. Twenty cats (20.6%) were haemoplasma positive; seven cats were infected only with Mycoplasma haemofelis, 10 were infected only with 'Candidatus Mycoplasma haemominutum' and three were co-infected with M haemofelis and 'Candidatus M haemominutum'. 'Candidatus Mycoplasma turicensis' was not detected. Haemoplasma infection was associated with older age (P=0.019). M haemofelis infection tended to be more common in anaemic cats (P=0.058). No association between gender and haemoplasma infection, or haemoplasma relative copy number and PCV, was detected. Retroviral infection rates were very low with only one FeLV proviral positive cat found.

Evaluation of real-time RT-PCR for the quantification of FCoV shedding in the faeces of domestic cats.

Faecal samples were taken from cats living in multi-cat households with endemic feline coronavirus (FCoV) infection. Total RNA was extracted from faecal suspensions and FCoV RNA was quantified using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The real-time RT-PCR threshold cycle (C(T)) values were consistently high suggesting that the samples contained very little viral RNA. However, experiments in which RNA extracted from FCoV-infected cell culture supernatants was combined with RNA extracted from faecal suspensions revealed the presence of faecal factors that significantly inhibited the reverse transcription reaction. Consequently, three methods of RNA extraction were investigated and RNA dilution was undertaken to investigate whether the effects of the faecal inhibitors could be reduced. Our results show that using the QIAgen RNA mini kit for RNA extraction and dilution of the RNA samples helps to reduce the inhibitory effects. However, because the extent of the inhibitory effects varied between faecal samples, accurate quantification proved difficult. We, therefore, conclude that although real-time RT-PCR provides an excellent method for detecting the presence of viral shedding, quantification of FCoV RNA in faecal material has to take into account the possible effects of RT-PCR inhibitors. It is, therefore, essential that all new assays, and the methods of sample preparation, are carefully evaluated before being used in a clinical setting.

Development of a strain-specific molecular method for quantitating individual campylobacter strains in mixed populations.

The identification of sites resulting in cross-contamination of poultry flocks in the abattoir and determination of the survival and persistence of campylobacters at these sites are essential for the development of intervention strategies aimed at reducing the microbial burden on poultry at retail. A novel molecule-based method, using strain- and genus-specific oligonucleotide probes, was developed to detect and enumerate specific campylobacter strains in mixed populations. Strain-specific oligonucleotide probes were designed for the short variable regions (SVR) of the flaA gene in individual Campylobacter jejuni strains. A 16S rRNA Campylobacter genus-specific probe was also used. Both types of probes were used to investigate populations of campylobacters by colony lift hybridization. The specificity and proof of principle of the method were tested using strains with closely related SVR sequences and mixtures of these strains. Colony lifts of campylobacters were hybridized sequentially with up to two labeled strain-specific probes, followed by the generic 16S rRNA probe. SVR probes were highly specific, differentiating down to 1 nucleotide in the target sequence, and were sufficiently sensitive to detect colonies of a single strain in a mixed population. The 16S rRNA probe detected all Campylobacter spp. tested but not closely related species, such as Arcobacter skirrowi and Helicobacter pullorum. Preliminary field studies demonstrated the application of this technique to target strains isolated from poultry transport crate wash tank water. This method is quantitative, sensitive, and highly specific and allows the identification and enumeration of selected strains among all of the campylobacters in environmental samples.

An update on FIV and FeLV test performance using a Bayesian statistical approach.

BACKGROUND: Screening tests for feline retroviruses are thought to have high sensitivity and specificity, although previous studies that evaluated these tests have limitations. Novel statistical approaches have been developed that allow the estimation of sensitivity and specificity in situations where the true state of the disease in individual animals cannot be assured. OBJECTIVE: The purpose of this study was to evaluate the sensitivity and specificity of a variety of retrovirus tests, including some screening tests, in a population of cats potentially infected with either feline leukemia virus (FeLV) and/or feline immunodeficiency virus (FIV) by using a Bayesian statistical approach. METHODS: Four hundred and ninety blood samples from cats being evaluated for FIV infection were tested by 2 rapid immunomigration tests (Witness single [WS], Witness combi [WC]) and a plate-based ELISA (Petcheck) for FIV antibody, and by a newly designed real-time polymerase chain reaction (PCR) assay for FIV provirus. Four hundred and ninety-five blood samples from cats being evaluated for FeLV infection were tested by 2 rapid immunomigration tests (WS, WC) and a plate-based ELISA (Petcheck) for FeLV antigen, and by a FeLV virus isolation technique. Results were then analyzed by using a Bayesian statistical method. RESULTS: For FIV tests, median sensitivity estimates were 0.98 for WS, 0.97 for WC, 0.98 for ELISA, and 0.92 for PCR. Median specificity estimates were 0.96 for WS, 0.96 for WC, 0.93 for ELISA, and 0.99 for PCR. For FeLV tests, median sensitivity estimates were 0.97 for WS, 0.97 for WC, 0.98 for ELISA, and 0.91 for virus isolation. Median specificity estimates were 0.96 for WS, 0.96 for WC, 0.98 for ELISA, and 0.99 for virus isolation. CONCLUSIONS: The use of Bayesian statistical methods overcomes a variety of methodologic problems associated with diagnostic test evaluations, including the lack of a definitive reference test. The sensitivity and the specificity of all 6 evaluated screening tests was high: however, specificity estimates were slightly lower than those reported by most recent studies.

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.