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BACKGROUND: Anaplasma phagocytophilum is the etiological agent of canine granulocytic anaplasmosis in dogs and causes human granulocytic anaplasmosis (HGA). Tick-borne anaplasmosis has been recognised as an emerging zoonotic health concern worldwide. The aim of the present study was to determine the prevalence of A. phagocytophilum in ticks collected from dogs in the UK and map its distribution. Routine surveillance of tick-borne disease is essential as part of a "One Health" approach to infectious disease management.METHODS: Tick DNA samples collected in 2015 as part of a large-scale tick surveillance programme were analysed using a previously validated diagnostic quantitative PCR for A. phagocytophilum.RESULTS: PCR analysis indicated that 138 out of 2994 tick DNA samples analysed were positive for A. phagocytophilum, a prevalence of 4.6% (95% CI: 3.89-5.42). Among these 138 tick DNA samples, 131 were from Ixodes ricinus, six were from Ixodes hexagonus and one was from Ixodes canisuga. Three of the I. ricinus tick DNA samples positive for A. phagocytophilum DNA were also positive for Borrelia spp. DNA and one was positive for Babesia spp. DNA, indicating co-infection. The ticks positive for the pathogen DNA were found widely distributed throughout the UK.CONCLUSIONS: These data provide important information on the prevalence and wide distribution of A. phagocytophilum in ticks infesting dogs within the UK.

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.

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.

Over 94 million domestic cats are considered pets, who, as our companions, are also susceptible to cancers, common and rare diseases. Whole exome sequencing (WES) is a cost-effective strategy to study their putative disease-causing variants. Presented is ~35.8 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. WES was conducted on 41 cats from various breeds with known and unknown diseases and traits, including 10 cats with prior whole genome sequence (WGS) data available, to test WES capture probe performance. A WES and WGS comparison was completed to understand variant discovery capability of each platform. At ~80x exome coverage, the percent of on-target base coverage >20x was 96.4% with an average of 10.4% off-target. For variant discovery, greater than 98% of WGS SNPs were also discovered by WES. Platform specific variants were mainly restricted to a small number of sex chromosome and olfactory receptor genes. Within the 41 cats with ~31 diseases and normal traits, 45 previously known disease or trait causal variants were observed, such as Persian progressive retinal degeneration and hydrocephalus. Novel candidate variants for polycystic kidney disease and atrichia in the Peterbald breed were also identified as well as a new cat patient with a known variant for cystinuria. These results show the discovery potential of deep exome sequencing to validate existing disease gene models and identify novel gene candidate alleles for many common and rare diseases in cats.

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.

In a large-scale survey in the UK, recruited veterinary practices were asked to inspect client-owned cats and dogs, selected at random between April and June 2018, following a standardised flea inspection protocol. A total of 326 veterinary practices participated and 812 cats and 662 dogs were examined during the 3-month period. Fleas were collected, identified to species level and fleas of the same species collected from a single animal were pooled together and treated as a single sample. A total of 470 pooled flea samples were screened by PCR and DNA sequence analysis for a subset of Rickettsia species including R. felis and R. typhi. On analysis, 27 (5.7%) of the pooled flea samples were positive for R. felis DNA; these were predominantly in the cat flea, Ctenocephalides felis, but one dog flea, Ctenocephalides canis was also positive for this pathogen.

The aim of this study was to investigate the clinical, pathologic, and viral etiology of horses with neurologic disorders. Twelve English Thoroughbred horses with neurologic disorders were investigated for the presence of neuropathogenic equine herpesvirus-1 (EHV-1) and Borna disease virus (BDV) by real-time polymerase chain reaction (PCR) and histopathological methods. Neuropathogenic EHV-1 was detected in the brain of two horses by real-time PCR. Borna disease virus p24 and p40 gene sequences were detected by real-time RT-PCR in the brain of a 3-year-old horse and in the blood of a 1-year-old horse. High fever, ataxia, depression, lack of coordination, and gait abnormalities were present in these horses, which died within a few days of developing neurologic signs. The BDV p24 and p40 real-time PCR products were sequenced and shown to be identical to previously reported BDV sequences. In the brain of the BDV-positive horse, hyperemia was pronounced in the parenchyma and the meninges. In addition, nonpurulent poliencephalomyelitis characterized by perivascular mononuclear cell infiltration was seen. Mononuclear and polymorphonuclear cell infiltration was also seen in the liver with necrotic perihepatitis besides subacute splenitis and glomerulonephritis and severe hyperemia in the kidney and edema and emphysema in the lung. In conclusion, neuropathogenic EHV-1 and BDV were detected in horses from Turkey using molecular detection methods. Since BDV genetic signatures were detected in Turkey for the first time, future epidemiologic studies need to be performed to investigate the range of host animals, spread, frequency, and molecular diversity of BDV; this will allow to determine the risk of this pathogen for Turkish veterinary and public health. © 2018 Elsevier Inc. 26675 The authors would like to thank to the University of Istanbul for funding this study (project no: 26675) and the Kansas Biosciences Authority. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either explicit or implicit, of the University of Kansas Biosciences Authority.

Background and Objective: Domestic animals, including camels, in Saudi Arabia suffer from various diseases, among which tick-borne infections are important because they reduce the productivity of these animals. However, knowledge of tick-borne pathogens in camels in Saudi Arabia is very limited, so the aims of this study were to quantify the abundance and distribution of tick species infesting camels from different districts of Riyadh province and use molecular tools to detect tick-borne pathogens in both the ticks and blood samples. Materials and Methods: A total of 218 ticks were collected from 116 camels from the 5 districts of Riyadh. The ticks and camel blood samples were analyzed for Borrelia, Babesia and Theileria pathogens using conventional and real-time PCR. Results: The results showed that five different tick species were identified. Majority of the ticks were Hyalomma dromedarii (70.6%), which were collected from camels in all 5 districts. This was followed by Hyalomma impeltatum species (25.2%), which was again found in all the districts. The other species found were Hyalomma anatolicum, Haemaphysalis sp. and Rhipicephalus turanicus. The only one H. dromedarii tick was positive for Theileria sp. DNA. Although the sample size and the area of tick collection were limited, the data suggest that the prevalence of pathogens in the Riyadh province, Saudi Arabia is relatively low. Conclusion: The study provides useful preliminary data to inform future full-scale country-wide surveys.

Background: Fleas (Siphonaptera) are the most clinically important ectoparasites of dogs and cats worldwide. Rising levels of pet ownership, climate change and globalisation are increasing the importance of a detailed understanding of the endemicity and prevalence of flea-borne pathogens. This requires continued surveillance to detect change. This study reports a large-scale survey of pathogens in fleas collected from client-owned cats and dogs in the UK.Methods: Recruited veterinary practices were asked to follow a standardised flea inspection protocol on a randomised selection of cats and dogs brought into the practice in April and June 2018. A total of 326 practices participated and 812 cats and 662 dogs were examined. Fleas were collected, identified to species and pooled flea samples from each host were analysed for the presence of pathogens using PCR and sequence analysis.Results: Overall, 28.1% of cats and 14.4% of dogs were flea infested. More than 90% of the fleas on both cats and dogs were cat fleas, Ctenocephalides felis felis. Fleas of the same species from each infested host were pooled. DNA was amplified from 470 of the pooled flea samples using conventional PCR, 66 of which (14% ± 95% CI 3.14%) were positive for at least one pathogen. Fifty-three (11.3% ± 95% CI 2.85%) of the pooled flea DNA samples were positive for Bartonella spp., 35 were from cats and 4 from dogs, the remainder had no host record. Seventeen of the Bartonella spp. samples were found to be Bartonella henselae, 27 were Bartonella clarridgeiae (of two different strains), 4 samples were Bartonella alsatica and one was Bartonella grahamii; 4 samples could not be identified. Fourteen (3% ± 95% CI 1.53%) of the flea DNA samples were found to be positive for Dipylidium caninum, 10 of the D. caninum-infected samples were collected from cats and one from a dog, the other 3 positive flea samples had no host species record. Only 3 flea samples were positive for Mycoplasma haemofelis or Mycoplasma haemocanis; 2 were collected from cats and one had no host species record. Three fleas were positive for both D. caninum and Bartonella spp. One flea was positive for both Bartonella spp. and M. haemofelis or M. haemocanis.Conclusions: This study highlights the need for ongoing flea control, particularly given the relatively high prevalence of Bartonella spp., which is of concern for both animal welfare and human health. The study demonstrates the ongoing need to educate pet owners about the effects of both flea infestation and also the pathogen risks these fleas present.

The outcome of infection with Leishmania infantum in dogs is variable, which is thought to be due to the nature of the immune response mounted by the host. As a consequence, the clinical signs and severity of canine leishmaniosis vary between individual dogs. Host immunogenetic factors might play an important role in determining the outcome of infection. The aim of this study was to examine polymorphisms in innate and adaptive immune response genes, to determine whether any of these were associated with susceptibility or resistance to L. infantum infection. Genomic DNA was obtained from two groups: pet dogs in endemic regions of Europe and a group of Beagles exposed to sand fly infection as part of a vaccine study. Genotyping was performed using a SNP (single nucleotide polymorphism) array for selected immune response genes. The first part of the study compared 62 clinical cases with 101 clinically unaffected dogs that were seronegative for Leishmania antibodies. One SNP in the CIITA gene demonstrated a significantly higher minor allele frequency in the case group, compared with the control group at the individual SNP level after permutation, but was not significant after correction for multiple testing. The second part of the study examined 48 Beagle dogs exposed to L. infantum over two transmission seasons. Twenty-seven dogs with a resistant phenotype (no evidence of clinical disease, seronegative at the end of the study period, negative on lymph node culture and only transiently PCR positive in bone marrow) were compared with 21 dogs demonstrating a susceptible phenotype (clinical disease, seropositive, positive lymph node culture and consistently PCR positive in bone marrow). Three SNPs in TLR3, two SNPs in PTPN22 and one SNP in TLR4 and IL1A were associated with the susceptible phenotype in the Beagle group at the individual SNP level after permutation analysis, but were not significant after correction for multiple testing. Further validation of these SNPs is required in a larger cohort of dogs, ideally with extreme phenotypes to confirm an association with the outcome of L. infantum infection.

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 (&chi, 2, p = 0.028), with a reduced risk of developing FIP (&chi, 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.

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Objectives The objectives of this study were to estimate the prevalence of feline haemoplasma infections in Northern Serbia, identify potential risk factors and perform molecular subtyping of feline immunodeficiency virus (FIV). Methods PCR analysis for feline haemoplasmas was performed on surplus EDTA blood samples from 373 cats from the Belgrade region, Serbia. An ELISA was used to determine the prevalence of feline leukaemia virus (FeLV) and FIV; PCR was performed on a subpopulation of these cats. FIV subtyping was performed using PCR. Results Within this population, 64/373 cats (17.2%) were infected with one or more haemoplasma species. Mycoplasma haemofelis was detected in 20/373 cats (5.4%), ‘ Candidatus Mycoplasma haemominutum’ in 47/373 cats (12.6%) and ‘ Candidatus Mycoplasma turicensis’ in 23/373 cats (6.2%). Coinfections were observed in 21/373 cats (5.6%). Based on ELISA serological retroviral testing, 4/310 cats (1.3%) were infected with FeLV, whereas 78/331 (23.6%) were infected with FIV. Multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio [OR] 2.7, 95% confidence interval [CI] 1.04–7.1; P = 0.041]), male gender (male/female, OR 4.5, 95% CI 2.22–9.03; P

Background: In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods: We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls.Results: From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis.Conclusions: Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR.

Background: Ticks derived from cats have rarely been evaluated for the presence of pathogens. The aim of this study was to determine the prevalence of Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks collected from cats in the UK. Methods: Five hundred and forty DNA samples extracted from 540 ticks collected from cats presenting to veterinarians in UK practices were used. Samples underwent a conventional generic PCR assay for detection of Hepatozoon spp. and real-time quantitative PCR assays for detection of Anaplasma phagocytophilum and three feline haemoplasma species and a generic qPCR for detection of Bartonella spp. Feline 28S rDNA served as an endogenous internal PCR control and was assessed within the haemoplasma qPCR assays. Samples positive on the conventional and quantitative generic PCRs were submitted for DNA sequencing for species identification. Results: Feline 28S rDNA was amplified from 475 of the 540 (88.0%) ticks. No evidence of PCR inhibition was found using an internal amplification control. Of 540 ticks, 19 (3.5%) contained DNA from one of the tick-borne pathogens evaluated. Pathogens detected were: A. phagocytophilum (n = 5; 0.9%), Bartonella spp. (n = 7; 1.3%) [including Bartonella henselae (n = 3; 0.6%) and Bartonella clarridgeiae (n = 1; 0.2%)], haemoplasma species (n = 5; 0.9%), "Candidatus Mycoplasma haemominutum" (n = 3; 0.6%), Mycoplasma haemofelis (n = 1; 0.2%), "Candidatus Mycoplasma turicensis" (n = 1; 0.2%), Hepatozoon spp. (n = 2; 0.4%), Hepatozoon felis (n = 1; 0.2%) and Hepatozoon silvestris (n = 1; 0.2%). Conclusion: These data provide important information on the prevalence of tick-borne pathogens in ticks infesting cats, with the identification of haemoplasma species, A. phagocytophilum, H. felis and Bartonella spp. (including B. henselae and B. clarridgeiae). This study also documents the first report of H. silvestris in ticks collected from domestic cats.

Feline infectious peritonitis (FIP) is a fatal disease of cats, and a sequela of systemic feline coronavirus (FCoV) infection. Mutations in the viral spike (S) gene have been associated with FCoVs found in tissues from cats with FIP, but not FCoVs found in faeces from healthy cats, and are implicated in monocyte/macrophage tropism and systemic spread. This study was designed to determine whether S gene mutation analysis can reliably diagnose FIP. Cats were categorised as with FIP (n = 57) or without FIP (n = 45) based on gross post-mortem and histopathological examination including immunohistochemistry for FCoV antigen. RNA was purified from available tissue, fluid and faeces. Reverse-transcriptase quantitative-PCR (RT-qPCR) was performed on all samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Samples were available from a total of 102 cats. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR-positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone. Electronic supplementary material The online version of this article (doi:10.1186/s13567-017-0467-9) contains supplementary material, which is available to authorized users.

Global importance: Hypokalaemic polymyopathy is a genetic disease of Burmese cats that has been encountered in Australasia, Europe and South Africa. Clinical features: Affected cats usually present with signs of muscle weakness and muscle pain in the first year of life. Although certain clinical features, such as ventroflexion of the head and neck, are especially characteristic, some cats do not display these signs. Usually weakness is periodic or episodic, but occasionally it is incessant. Diagnostic challenges: In the past, diagnosis was problematic in that clinical signs and a lowered serum potassium concentration were not always observed synchronously. This necessitated serial serum potassium concentration determinations, testing of serum creatine kinase activity and exclusion of other potential causes of muscle disease in cats (including muscular dystrophies, Toxoplasma myositis, immune-mediated polymyositis, organophosphorus intoxication and envenomations). Signs in affected cats often waxed and waned, possibly in response to changes in dietary factors and stress, and some cats could apparently ‘grow out of’ the condition. Recent advances and future prospects: Recent molecular genetics research has identified a single nonsense mutation in the gene ( WNK4) coding for lysine-deficient 4 protein kinase, an enzyme present primarily in the distal nephron. The underlying pathomechanism in affected cats is therefore likely to be a potassium wasting nephropathy, as this enzyme is involved in complex sodium/potassium exchange mechanisms in the kidney. Additional functional characterisation of the condition is warranted to define precisely how, why and when the serum potassium concentration declines. The diagnosis of Burmese hypokalaemia is now straightforward, as an inexpensive PCR test can identify affected homozygous individuals, as well as carriers. The elimination of this condition from the Burmese breed, and also from pedigree cats infused with Burmese lines, such as the Bombay, Tonkinese and Tiffanie breeds, should therefore be possible.

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.

In a study of tick and tick-borne pathogen prevalence, between May and October 2016, 278 veterinary practices in Great Britain examined 1,855 cats. Six-hundred and one cats were found to have attached ticks. The most frequently recorded tick species was Ixodes ricinus (57.1%), followed by Ixodes hexagonus (41.4%) and Ixodes trianguliceps (1.5%). Male cats, 4 − 6 years of age living in rural areas were most likely to be carrying a tick; hair length and tick treatment history had no significant association with attachment. For cats that were parasitized by ticks in large urban areas, I. hexagonus was the most frequent species recorded. Molecular analysis was possible for 541 individual tick samples, others were too damaged for analysis; Babesia spp., and Borrelia burgdorferi sensu lato were identified in 1.1% (n = 6) and 1.8% (n = 10) of these, respectively. Babesia spp. included Babesia vulpes sp. nov./Babesia microti-like (n = 4) in I. hexagonus and Babesia venatorum (n = 2) in I. ricinus. Borrelia burgdorferi s.l. species included Borrelia garinii (n = 6) and Borrelia afzelii (n = 4). The majority of B. burgorferi s.l. cases were found in I. ricinus, with B. afzelii in one I. hexagonus nymph. No Borrelia or Babesia spp. were present in I. trianguliceps. To determine a true prevalence for ticks on cats, practices that only submitted questionnaires from cats with ticks and practices that submitted fewer than 5 returns per week were removed; amongst those considered to have adhered strictly to the collection protocol, feline tick prevalence amongst cats that had access to the outdoors was 6.6%. These results show that ticks can be found on cats throughout Great Britain, which harbour a range of species of Babesia and B. burgdorferi s.l. and that cats, particularly in green spaces within urban areas, may form an important host for I. hexagonus, a known vector of pathogens.

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management.

Background Feline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus. Methods A cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), “Candidatus Mycoplasma haemominutum” (CMhm) and “Candidatus Mycoplasma turicensis” (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents. Results Ninety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt infection. Conclusions A high prevalence of infectious agents was found in cats from Cyprus with Mhf, CMhm, CMt, L. infantum, B. henselae, H. felis, A. platys, FeLV and FIV infections reported for the first time. The significant associations between different pathogens provide a better understanding of similarities in the epidemiology of these pathogens and interactions between them. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2063-2) contains supplementary material, which is available to authorized users.

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.

Objectives The aim of the study was to investigate feline morbillivirus (FmoPV) frequency, phylogeny and associated pathology in cats in Istanbul, Turkey. Methods Samples from sick (n = 96) and dead ( n = 15) cats were analysed using reverse transcription PCR. Blood and urine analyses and histopathology were also performed. Results FmoPV RNA was detected in six cats (5.4%), including three sick (in the urine) and three dead cats (tissues). A significantly greater proportion of FmoPV RNA-positive cats had street access compared with non-infected cats. Blood samples from the morbillivirus-positive cats were negative for morbillivirus RNA. Tubular parenchymal cells, lymphoid and plasma cells in kidney and hepatocytes, lymphoid and plasma cells in liver from dead cats were also positive by immunohistochemistry for the viral N protein. Two FmoPV-positive cats were also positive for feline coronavirus RNA and one cat for feline immunodeficiency virus RNA and feline leukaemia virus proviral DNA. Phylogenetic analysis of the six FmoPV-positive cats showed that the strains were grouped into cluster D and had high similarity (98.5–100%) with strains from Japan and Germany. In the three FmoPV RNA-positive sick cats, respiratory, urinary and digestive system signs were observed as well as weight loss, fever and depression in some cats. Similar clinical signs were also seen in the morbillivirus RNA-negative sick cats. FmoPV RNA-positive cats had lower median red blood cell count, haemoglobin, albumin, albumin/globulin and urobilinogen and higher alanine transaminase, alkaline phosphatase and bilirubin compared with non-infected cats. Significant histopathology of FmoPV RNA-positive dead cats included tubulointerstitial nephritis characterised by severe granular and vacuolar degeneration of the epithelial cells of the cortical and medullary tubules as well as mononuclear cell infiltrates. Widespread lymphoid cell infiltrates were detected in the renal cortex and medullary regions of the kidneys. Cellular infiltration, cholangiohepatitis and focal necrosis in the liver were also found. Although virus-infected cells were found in the kidney and liver of FmoRV RNA-positive cats, tubulointerstitial nephritis, cholangiohepatitis and focal necrosis seen in FmoRV RNA-positive cats were similar to those observed in FmoRV RNA-negative cats. Conclusions and relevance This is the first study to show the presence of FmoPV infection in cats in Turkey. Sick cats, particularly those with kidney disease, should be tested for this virus. The genotypes found in this study were similar to previously reported strains, indicating that circulating morbilliviruses in Turkey are conserved.

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.

OBJECTIVES To determine prevalence and demographics of two myosin-binding protein C (MYBPC3) mutations that affect ragdolls (R820W) and Maine coons (A31P) in the British Isles. METHODS From the database of a genetic testing laboratory samples from 2018 ragdolls and 742 Maine coons were analysed with respect to mutation status, age, sex and county of origin. The actual prevalence was compared to the expected Hardy–Weinberg prevalence by chi-squared test. RESULTS The prevalence of the R820W mutation in ragdolls was 27% (25·6% heterozygous, 1·4% homozygous), and that of the A31P mutation in Maine coons was 39·4% (36·4% homozygous, 3% heterozygous). There were more female cats (69·5% ragdoll, 70·3% Maine coon). The median age was 6·4 months (ragdolls) and 5·9 months (Maine coons). Cats from more than 60 counties were represented for each breed. The difference between the expected and observed allele frequency was significant in Maine coons (P=0·047) but not in ragdolls (P=0·092). CLINICAL SIGNIFICANCE This is the first report of prevalence and demographics of the R820W and A31P mutations in ragdolls and Maine coons, respectively, in the British Isles. The prevalence is high, which is of relevance for breeding and screening programmes. The significant difference in genetic distribution may suggest early death of homozygous Maine coons.

Background Feline coronavirus (FCoV) infection is common. In a small percentage of cats, FCoV infection is associated with the fatal disease feline infectious peritonitis (FIP). Genetically distinct virulent and avirulent strains of FCoV might coexist within a cat population. Objectives To determine whether the strains of FCoV in FIP-affected cats are closely related or genetically distinct from the fecally derived strains of FCoV in contemporary-asymptomatic cats during an epizootic outbreak of FIP. Animals Four cats euthanized because of FIP and 16 asymptomatic cats. Methods This prospective outbreak investigation was initiated during an outbreak of FIP in cats within or rehomed from a rescue/rehoming center. Postmortem samples were collected from cats with FIP and contemporaneous fecal samples from asymptomatic cats. RNA was purified from tissue and fecal samples, FCoV gene fragments were reverse transcribed, PCR-amplified using novel primers, and sequenced. Sequences were aligned with ClustalW and compared with published FCoV sequences. Results FCoV RNA was detected in all 4 FIP cat postmortem samples and in 9 of the 16 fecal samples from contemporary-asymptomatic cats. Novel primers successfully amplified fragments from 4 regions of the genome for all FCoV-positive samples. Phylogenetic analysis showed that the FIP-associated strains of FCoV from the outbreak were very closely related to the fecally derived strains of FCoV from contemporary-asymptomatic cats. Conclusions and Clinical Importance Sequence analysis provided no evidence that genetically distinct virulent and avirulent strains of FCoV were present during this FIP outbreak.

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.

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.

The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase is associated with the production of sialic acids on cat red blood cells. The cat has one major blood group with three serotypes; the most common blood type A being dominant to type B. A third rare blood type is known as AB and has an unclear mode of inheritance. Cat blood type antigens are defined, with N-glycolylneuraminic acid being associated with type A and N-acetylneuraminic acid with type B. Blood type AB is serologically characterized by agglutination using typing reagents directed against both A and B epitopes. While a genetic characterization of blood type B has been achieved, the rare type AB serotype remains genetically uncharacterized. A genome-wide association study in Ragdoll cats (22 cases and 15 controls) detected a significant association between blood type AB and SNPs on cat chromosome B2, with the most highly associated SNP being at position 4,487,432 near the candidate gene cytidine monophospho-N-acetylneuraminic acid hydroxylase. A novel variant, c.364C>T, was identified that is highly associated with blood type AB in Ragdoll cats and, to a lesser degree, with type AB in random bred cats. The newly identified variant is probably linked with blood type AB in Ragdoll cats, and is associated with the expression of both antigens (N-glycolylneuraminic acid and N-acetylneuraminic acid) on the red blood cell membrane. Other variants, not identified by this work, are likely to be associated with blood type AB in other breeds of cat.

Objectives: The aims of this study were to determine the prevalence of canine haemoplasmas, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” infection in Central Macedonia, Greece, and to evaluate any associations between canine haemoplasma infection and clinical presentation, selected laboratory data or the presence of ticks. Methods: Genomic DNA was purified from excess blood (n=151) submitted for haematological examination. Purified DNA was subjected to species-specific quantitative polymerase chain reaction assays duplexed with a canine DNA control quantitative polymerase chain reaction. Clinical records were retrospectively examined and selected clinical parameters were compared to haemoplasma infection status. Results: Nine samples were excluded due to inadequate canine DNA polymerase chain reaction results. Of the remaining 142 samples: eight (5·6%) were positive for M. haemocanis alone, six (4·2%) were positive for “Ca. M. haematoparvum” alone and one (0·7%) was dual positive. No association was found between haemoplasma status and age, sex, breed, health status, presence of anaemia, selected biochemistry parameters, presence of ectoparasites, routine ectoparasiticide treatment or the presence of selected tick-borne diseases.

Purpose To investigate the possible inhibition of qPCR assays used for the diagnosis of ocular infections in cats by proxymetacaine, fluorescein, and fusidic acid, which are commonly used in veterinary ophthalmology. Methods Fluorescein, proxymetacaine, and fusidic acid were tested for possible inhibition of a triplex qPCR assay designed to detect Chamydophila felis, Feline herpesvirus 1 (FHV-1), and the feline 28S ribosomal DNA (28S rDNA) gene by comparing threshold cycle (Ct) values of samples with and without the three products. A second experiment was carried out to measure the effects of various dilutions of fusidic acid. Results No statistically significant differences were detected between the C. felis, FHV-1, and 28S rDNA Ct values with and without proxymetacaine or fluorescein. However, there was a statistically significant increase in FHV-1 (P

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.

The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

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.

Objective: The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. Methods: Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results: Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance: Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.

The aim of the present study was to characterize the antigenic specificity of the humoral immune response made by cats infected with the feline hemoplasma,Mycoplasma haemofelis.A crudeM. haemofelisantigen preparation was prepared from red blood cells (RBCs) collected from a cat at the time of a high level of bacteremia. Plasma samples were collected from six cats before and after experimental infection withM. haemofelis, with regular sampling being performed from 15 to 149 or 153 days postinfection (dpi). Preinfection RBC membrane ghosts were prepared from these six cats and used to identify erythrocyte proteins that may have contaminated theM. haemofelisantigen preparation. TheM. haemofelisantigen preparation comprised 11 protein bands. The immunodominant bands on Western blotting with infected cat plasma had molecular masses of 78, 68, 60, 48, and 38 kDa. Most cats (n= 5) had plasma antibody that reacted with at least one band (always including the one of 68 kDa) at 15 dpi, and all cats were seroreactive by 29 dpi. The maximum number of antibodies from an individual animal specific for an antigen was identified in plasma collected from 57 to 99 dpi. Contamination of theM. haemofelisantigen preparation with RBC membrane proteins was observed. The contaminating RBC proteins had molecular masses of from 71 to 72 kDa (consistent with band 4.2) and 261 and 238 kDa (consistent with spectrin), and these were recognized by all plasma samples. A range ofM. haemofelisantigens is recognized by cats infected experimentally with the organism. These represent possible targets for immunoassays, but care must be taken to prevent false-positive results due to host protein contamination.

Chlamydophila felis is a common cause of conjunctivitis in cats. Greater understanding of C. felis infection and immunity and identification of protective antigens will facilitate improved vaccine design. Chlamydial polymorphic membrane proteins (Pmps) represent a family of homologous proteins of likely importance in chlamydial infection and immunity. To identify immunogenic C. felis Pmps, we generated recombinant C. felis Pmps (rPmps) and used these to detect serum antibody reactivity against Pmps arising during C. felis infection in cats. Sequencing of Pmp genes 1, 7, 13, 18, 19 and 20 from 3 laboratory strains of C. felis (K2487, 1497V and Cello) and alignment with the Fe/C-56 genome revealed high genetic identity in Pmp genes between strains. PCR products lacking the predicted N-terminal signal sequence peptide and C-terminal domain were generated, cloned and expressed in Escherichia coli prior to purification by nickel-agarose affinity chromatography. Serum samples from 4 cats collected up to 55 days post-inoculation with C. felis (K2487) were analysed by western blotting and rPmp-specific ELISAs for evidence of serum antibody reactivity against each rPmp. Strong serum antibody reactivity against rPmps 1 and 7, and weak heterogeneous serum immunoreactivity against rPmps 13, 19 and 20, were detected from 14 to 21 days post-infection (dpi), peaking at 28-35 dpi and tending to plateau thereafter. No significant serum antibody reactivity was detected against rPmp18. This study provides the first evidence that C. felis Pmps 1 and 7 are likely to represent immunodominant proteins and recommends investigation of their potential as serodiagnostic antigens and novel vaccine candidates.

Mycoplasma haemocanis is a hemotropic bacterium that can be associated with acute hemolytic disease in immunocompromised or splenectomized dogs. The present case report describes for the first time the use of real-time quantitative polymerase chain reaction (qPCR) to monitor M. haemocanis infection in a splenectomized dog. The report also describes the application of real-time qPCR for the analysis of deoxyribonucleic acid extracted from stained blood films. The analysis of blood films from the time of initial presentation allowed a retrospective confirmation of M. haemocanis infection. The M. haemocanis copy numbers remained high throughout antibiotic treatment of this dog. A decline in copy numbers was only recorded after 11 months of therapy, when improvements in clinical and hematological indices were also noted. Clearance of infection was not achieved, and the dog remained positive for M. haemocanis at 3.5 months postcessation of antibiotic therapy. Cytological examination of blood films for the presence of organisms was insensitive for the detection of parasitemia.

[This corrects the article DOI: 10.1371/journal.pone.0154973.].

Norovirus (NoV) is recognised as one of the most common causes of foodborne infections, and shellfish are a well-documented source of this virus. The presence of NoV in shellfish has not previously been investigated in Turkey, and hence the aim of this study was to determine the frequency of human NoV genogroups I and II in mussels collected from the Bosphorus, Istanbul, Turkey. A total of 320 mussels representing 110 samples originating along the Bosphorus coast were collected from fish distributors. RNA was extracted using the RNeasy Mini Kit and real-time RT-PCR performed using primers specific for NoV genogroup I and II. Amongst the 110 samples, 5 (4.5%) were found to be positive for NoV genogroup II by SYBR Green assay; no genogroup I was detected. A positive signal was obtained by SYBR Green for NoV Genogroup II in mussels collected in October, November and December 2008, and February and July 2009. Only four out of five SYBR Green positive samples could be confirmed by the use of a NoV GII probe-based real-time RT-PCR. The average count and SD of Enterobactericaeae, E. coli and sulphide reductase anaerobic bacteria in PCR positive mussels were 3.56 log +/- 0.96 log, 2.32 log +/- 0.77 log and 1.70 log +/- 0.56 log, respectively. This study shows that NoV Genogroup II is present in mussels collected from the Bosphorus, Istanbul, and may constitute a risk to human health.

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185). QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 106 copies of the sequence-specific plasmid from the non-target canine haemoplasma species. Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected. This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.

The aim of the study was to describe blood and tissue copy number distribution during Mycoplasma haemofelis infection and determine if sequestration of organisms in body tissues could explain blood copy number cycling in infected cats. Thirteen domestic–shorthaired cats were used. Blood samples were regularly collected, and at a differing time point post-infection for each cat, tissue samples also collected, for quantitative PCR (qPCR). Absolute haemoplasma copy numbers were calculated for all blood and tissue samples, as well as an estimation of the ratio of tissue haemoplasma copy number to that expected in the tissue if a positive qPCR result arose due to tissue blood supply alone. Cats with high or moderate M. haemofelis blood copy numbers at the time of tissue collection had fewer M. haemofelis copies in most tissues than expected due to the tissue blood supply alone; only splenic and lung tissues consistently contained more M. haemofelis. However tissues collected from cats at a time of very low M. haemofelis blood copy numbers, when putative copy number cycling nadirs were occurring, were usually qPCR negative. Hence no evidence of significant tissue M. haemofelis sequestration was found in this study to explain the copy number cycling reported with this feline haemoplasma species.

The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), 'Candidatus M. haemominutum' (Group HM: 3 cats) or 'Candidatus M. turicensis' (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs' testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P