Detection of feline haemoplasma species in experimental infections by in-situ hybridisation.

The aim of this study was to use fluorescence in-situ hybridisation (FISH) to search for the tissues and cell types important in survival and persistence of Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum" or "Candidatus Mycoplasma turicensis" in infected cats. A 16S rDNA probe for each species was applied to formalin-fixed, paraffin wax-embedded tissues sections collected from experimentally infected cats. Tissues (n = 12) were collected, at necropsy, from ten cats which had been infected with M. haemofelis, and one each with "Ca. M. haemominutum" and "Ca. M. turicensis". M. haemofelis specific hybridisation was present on red blood cells (RBCs) in all tissues from acutely infected cats, but not the majority of tissues from chronically infected cats. "Ca. M. haemominutum" specific hybridisation was present on scattered RBCs within the spleen and liver. Specific probe hybridisation was not detected in any of the "Ca. M. turicensis" infected tissues. Haemoplasmas were detected on the surface of RBCs only and not any other cell type. Additionally, FISH was limited by sensitivity and could not detect the lower numbers of organisms present in tissues of cats chronically infected with M. haemofelis. Occasional organisms were detected in cats acutely infected with "Ca. M. haemominutum" but not "Ca. M. turicensis".

Distribution of Mycoplasma haemofelis in blood and tissues following experimental infection.

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.

Alternaria species infection in nine domestic cats.

A case series of nine domestic cats with culture-confirmed Alternaria species infection is presented, with conclusions drawn regarding signalment, clinical signs, treatment and outcome. Middle aged neutered males were over-represented and all presented with cutaneous lesions involving the extremities (nose, pinnae and digits). Lesions were mainly slow-growing, poorly circumscribed nodules or plaques but some also presented as non-healing wounds. A combination of surgical excision with adjunctive medical therapy appeared to be the most successful treatment option but long courses of medical therapy were generally required and recurrence was common.

Risk factors for feline immunodeficiency virus antibody test status in Cats Protection adoption centres (2004).

A study was carried out to determine the prevalence of feline immunodeficiency virus (FIV) within a population of cats entering 10 UK adoption centres run by Cats Protection. All cats entering the adoption centres during 2004 were tested for FIV using a rapid enzyme immunoassay antibody test. The overall prevalence of positive test results was 3.1% (95% confidence intervals (CI) 2.7-3.5%), whilst the prevalence at different adoption centres varied from 0.8% (95% CI 0.1-1.5%) to 6.7% (95% CI 4.9-8.5%). Results of the multivariable logistic regression analysis showed that male cats, stray/feral cats and cats in poor health were at a greater risk of testing positive for FIV than female cats, cats that were relinquished by an owner and cats that were in good/fair health, respectively. No evidence was found for an association between neuter status and FIV test results. This study may help to identify cats that are relinquished to rescue centres with an increased risk of FIV for routine FIV testing.

Field study assessing the performance of a patient-side blood test to determine neuter status in female cats based on detection of luteinising hormone.

OBJECTIVES: The aim of this study was to assess the performance of a patient-side blood test in determining neuter status in female cats. METHODS: Residual blood samples from female cats of unknown neuter status that were admitted to four cat adoption centres in the UK were tested for luteinising hormone (LH) using the Witness LH test (Zoetis). A positive LH test result indicated that the cat was neutered. Cats were assessed for evidence of a surgical scar suggestive of prior neutering; if none was found, an exploratory laparotomy was performed to confirm neuter status. The LH test performance was assessed (sensitivity, specificity, negative and positive predictive value). RESULTS: Two hundred and thirty-six cats had both LH test and exploratory laparotomy data. The specificity of the test in detecting neutered cats was 100% (95% confidence interval 96.2-99.9) and the sensitivity was 69% (95% confidence interval 59.3-76.8). The prevalence of neutered cats in this sample was 49%. The positive and negative predictive values were 1 and 0.77, respectively. CONCLUSIONS AND RELEVANCE: The Witness LH test correctly detected all unneutered cats and thus there were no false-positive results that incorrectly indicated a cat was neutered. This study therefore suggests that positive LH test results avoid the need to perform surgery to confirm neuter status. This has significant welfare benefits for cats as it provides a lower risk, faster and less traumatic alternative to surgery and, in the shelter setting, it will have a positive impact on the cost, speed of assessment and time to rehoming of cats.

RNase P RNA gene (rnpB) phylogeny of Hemoplasmas and other Mycoplasma species.

Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and other Mycoplasma species. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related to Mycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.

A study of risk factors for cat mortality in adoption centres of a UK cat charity.

A case-control study was used to identify variables associated with the risk of mortality in cats housed at adoption centres. Multivariable logistic regression, based on retrospective data collected for 194 cases (cats that died or were euthanased) and 320 controls (cats that did not die) revealed an increased risk of mortality for cats admitted to adoption centres unneutered, in fair/poor health and cats born at adoption centres. Cats aged 7 weeks or less and cats aged over 7 years had an increased risk of mortality compared with cats of other ages. The risk of mortality decreased as the time in the adoption centre increased. Cats with disabilities (eg, blindness) had a higher mortality risk than cats without disabilities. Knowledge of these risk factors can inform intervention strategies aimed at reducing the risk of cat mortality at adoption centres.

Use of real-time PCR to detect Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in the saliva and salivary glands of haemoplasma-infected cats.

Feline haemoplasma infection can cause haemolytic anaemia. The natural method of transmission of haemoplasmas between cats is currently unknown but the nature of some of the risk factors for infection suggests that saliva may act as a mode of transmission. The aim of this study was to determine if Mycoplasma haemofelis (Mhf) and 'Candidatus Mycoplasma haemominutum' (CMhm) DNAs could be amplified from saliva and salivary gland samples collected from haemoplasma-infected cats.

Owner-reported lower urinary tract signs in a cohort of young cats.

Objectives The most common cause of lower urinary tract signs (LUTS) in cats under the age of 10 years is feline idiopathic cystitis (FIC). The prevalence of LUTS in the UK pet cat population is difficult to assess. This study used data collected prospectively to investigate the prevalence of, and risk factors for, owner-reported LUTS in a cohort of young pet cats. Methods Cat owners were recruited into a long-term longitudinal study and asked to complete questionnaires at specified age points for their cats. All cats were at least 18 months of age at the time of analysis. The prevalence of owner-reported LUTS at 18, 30 and 48 months of age was calculated, based on whether the owner had seen the cat urinating, and whether the cat had displayed one or more of the following clinical signs: dysuria, haematuria or vocalising during urination. A case-control study to investigate the risk factors for owner-reported LUTS in study cats at age 18 months was also conducted, using a multivariable logistic regression model. Results The prevalence of owner-reported LUTS in cats seen urinating by the owner was 4.3%, 3.8% and 6.0%, with 95% confidence intervals of 3.2-5.7%, 2.5-5.7% and 3.4-10.5% at ages 18, 30 and 48 months, respectively. An indoor-only lifestyle at the age of 18 months and a change in diet between the ages of 12 and 18 months were identified as risk factors for owner-reported LUTS at the age of 18 months from the multivariable model. No clear type of change in diet was identified in our sample of cats with LUTS. Conclusions and relevance The prevalence of owner-reported LUTS in a cohort of young pet cats was higher than the previously reported prevalence of LUTS in cats presenting to veterinary hospitals for LUTS or other reasons. A novel risk factor of change in diet between 12 and 18 months of age warrants further investigation.

Effect of chronic feline immunodeficiency infection, and efficacy of marbofloxacin treatment, on 'Candidatus Mycoplasma haemominutum' infection.

The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on 'Candidatus Mycoplasma haemominutum' infection. Six cats chronically infected with FIV-Glasgow8 (group A) and six FIV-free cats (group B) were infected with 'Candidatus M. haemominutum' on day 0 by intravenous inoculation of blood. From day 0 to 105 post-infection (pi), blood samples were collected for 'Candidatus M. haemominutum' and FIV provirus quantitative real-time polymerase chain reaction (PCR) and haematological examination. Three of the six cats in each of the groups were randomly selected to receive marbofloxacin treatment (2mg/kg PO SID) from day 49 to day 76 pi, with the remaining cats being untreated controls. Maximum 'Candidatus M. haemominutum' copy number was reached around day 30 pi. No overt cycling or marked variation in copy number was observed. No significant effect of FIV infection on 'Candidatus M. haemominutum' copy number kinetics or anaemia indices was found. No correlation was found between FIV provirus copy number and 'Candidatus M. haemominutum' copy number or haematological variables. Although marbofloxacin treatment was associated with a significant decrease in 'Candidatus M. haemominutum' copy number, the copy number plateaued during treatment, with no negative PCR results. Additionally, after termination of marbofloxacin treatment the copy numbers of the treated cats increased to reach levels similar to those of the untreated cats within 7-10 days. This study documents, for the first time, the infection kinetics and antibiotic responsiveness of 'Candidatus M. haemominutum' infection.

Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection.

The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Six cats chronically infected with FIV-Glasgow8 (Group X) and six FIV-free cats (Group Y) were infected with M. haemofelis on Day 0 by intravenous blood inoculation. From Day 0 until Day 86 post-infection (pi), blood samples were collected for M. haemofelis and FIV provirus quantitative real-time PCR and haematology. Three of the six cats in each of Groups X and Y were randomly selected to receive marbofloxacin treatment (2 mg/kg PO q24 h) from Day 16 to 43 pi, with the remaining cats being untreated controls with no antibiotic treatment. The M. haemofelis copy numbers and haematological data were compared between Groups X and Y, and between marbofloxacin-treated and control cats using a Mann-Whitney U-test. M. haemofelis infection was associated with development of macrocytic hypochromic anaemia. In some cats, marked variation in M. haemofelis copy number over time (>100,000-fold difference within 48 h in some cats) and/or cycling of copy number was seen. No correlation was found between FIV provirus copy number and M. haemofelis copy number or haematological variables. No significant effect of chronic FIV infection on M. haemofelis copy number kinetics or haematological changes due to M. haemofelis infection was found, other than MCHC (P=0.03). Marbofloxacin treatment was associated with a significant decrease in M. haemofelis copy number (P=0.002), although consistent clearance of infection was not demonstrated. This study reveals the presence of marked fluctuations in M. haemofelis copy number kinetics in vivo and a significant response to marbofloxacin antibiotic treatment.

A questionnaire-based study of gestation, parturition and neonatal mortality in pedigree breeding cats in the UK.

This study was based on a convenience-sampling questionnaire study of pedigree cat breeding in the UK. Data were collated for the births of 1,056 litters from 14 different pedigree breeds and 942 different households. Significant relationships between various outcomes and relevant predictors were assessed by multiple linear regression or logistic regression as appropriate. The overall mean gestation length of 65.1 days varied significantly between the breeds (P<0.0001), and larger litter sizes were associated with shorter gestation lengths (P=0.04). The mean litter size of 4.6 kittens also varied significantly according to breed (P<0.0001). The weight of kittens born alive (overall mean 93.5 g) increased with longer gestation lengths (P=0.0003), decreased with larger litter sizes (P<0.0001) and varied between the breeds (P<0.0001). A total of 8.0% of pregnancies resulted in a caesarean section, with a higher risk associated with smaller litter sizes (P=0.002). Although the frequency of caesarean sections varied from 0 to 18.5% between individual breeds, breed itself was not shown to have a significant independent effect on this likelihood. A mean of 7.2% of all the kittens were stillborn, which varied according to breed (P=0.0003), and the risk of a stillborn kitten increased with litter size (P=0.0001), and with the presence of congenital defects in the litter (P=0.0002). The mean kitten mortality between birth and 8 weeks of age was 9.1%, and the majority of these occurred in the first week of life. Parturition intervals varied widely. The duration of first stage of labour was less than 2h in 82.9% of cats. The interval between the birth of the first and last kitten was less than 6h in 85.7%, but more than 48 h in three cats. A maximum of 48 h was recorded between the births of individual kittens in unassisted deliveries.

Use of a Taqman PCR to determine the response of Mycoplasma haemofelis infection to antibiotic treatment.

A quantitative Taqman polymerase chain reaction (PCR) assay was used to evaluate the response of Mycoplasma haemofelis experimentally infected cats to three antibiotic treatment regimes. Sixteen cats were intravenously inoculated with M. haemofelis from a chronically infected donor. The cats were randomly assigned to one of four treatment groups each containing four cats: oral doxycycline at 10 mg/kg/day for 14 days, oral enrofloxacin at 5 mg/kg/day for 14 days, oral enrofloxacin at 10 mg/kg/day for 14 days, and an untreated control group. DNA, extracted from blood samples collected on days 0, 7, 14, 21, 25, 28, 32, 35, 42 and 54 post-inoculation (PI), was subjected to quantitative Taqman PCR. The M. haemofelis copy number was significantly lower in the doxycycline group (P=0.008), the 5 mg/kg/day enrofloxacin group (P=0.006) and the 10 mg/kg/day enrofloxacin group (P=0.005) compared to the untreated control group. No significant differences were found between any of the three antibiotic treated treatment groups. All three antibiotic treatment regimes evaluated in this study were effective at reducing M. haemofelis copy number.

Immune cell populations in the duodenal mucosa of cats with inflammatory bowel disease.

The aim of this study was to characterize the phenotype of leukocytes infiltrating the duodenal mucosa of cats with inflammatory bowel disease (IBD) by using immunohistochemistry and computer-aided morphometry to assess whether immunologic markers would aid in characterization of IBD. Frozen and formalin-fixed duodenal biopsies were collected from cats referred for investigation of chronic vomiting, diarrhea, or both (n = 34). Reference ranges were previously established by using duodenal samples from healthy cats (n = 16). No significant difference was found in the number of immunoglobulin G+ (IgG+) or IgA+ in either the villous lamina propria or the crypt lamina propria between cats with IBD and control cats. T cells (CD3+) increased in number from crypt to the tip of the villi in biopsies from both diseased (mean +/- SD for each group was 18.8 +/- 6.6 and 17.7 +/- 4.2 cells/ 10,000 m2 in cryptal areas to 25.2 +/- 9.5 and 29.1 +/- 13.3 cells/10,000m2 in villous areas) and healthy animals (17.9 +/- 3.9 cells/10,000 microm2 in cryptal areas to 24.1 +/- 9.3 cells/10,000 microm2 in villous areas) and no significant difference was found between diseased and control cats. By contrast, major histocompatibility complex (MHC) class II expression by leukocytes with dendritic cell or macrophage morphology in the lamina propria was significantly greater in cats with IBD (13.3 +/- 4.2 cells/10,000 microm2 in cryptal area; P = .016) than in healthy cats (11.9 +/- 3.0 cells/10,000 microm2) and MHC class II expression by enterocytes also was more pronounced in these cats showing an overall intensity of expression of 7.1 +/- 4.0 cells/10,000 microm2 in cats with IBD as opposed to 0.0 +/- 0.0 cells/10,000 microm2 to 0.3 +/- 0.7 cells/10,000 microm2 in healthy cats. These findings suggest that a subtle immunologic dysregulation occurs in spontaneously arising feline IBD.

Extent of linkage disequilibrium in the domestic cat, Felis silvestris catus, and its breeds.

Domestic cats have a unique breeding history and can be used as models for human hereditary and infectious diseases. In the current era of genome-wide association studies, insights regarding linkage disequilibrium (LD) are essential for efficient association studies. The objective of this study is to investigate the extent of LD in the domestic cat, Felis silvestris catus, particularly within its breeds. A custom illumina GoldenGate Assay consisting of 1536 single nucleotide polymorphisms (SNPs) equally divided over ten 1 Mb chromosomal regions was developed, and genotyped across 18 globally recognized cat breeds and two distinct random bred populations. The pair-wise LD descriptive measure (r(2)) was calculated between the SNPs in each region and within each population independently. LD decay was estimated by determining the non-linear least-squares of all pair-wise estimates as a function of distance using established models. The point of 50% decay of r(2) was used to compare the extent of LD between breeds. The longest extent of LD was observed in the Burmese breed, where the distance at which r(2) ≈ 0.25 was ∼380 kb, comparable to several horse and dog breeds. The shortest extent of LD was found in the Siberian breed, with an r(2) ≈ 0.25 at approximately 17 kb, comparable to random bred cats and human populations. A comprehensive haplotype analysis was also conducted. The haplotype structure of each region within each breed mirrored the LD estimates. The LD of cat breeds largely reflects the breeds' population history and breeding strategies. Understanding LD in diverse populations will contribute to an efficient use of the newly developed SNP array for the cat in the design of genome-wide association studies, as well as to the interpretation of results for the fine mapping of disease and phenotypic traits.

Proportion of pet cats registered with a veterinary practice and factors influencing registration in the UK.

Registration of a cat with a veterinary practice is likely to be a critical factor for access to key preventative medicine. A cross-sectional study was conducted to collect data in the United Kingdom on the registration status of cats and potential explanatory variables. These data were also used to identify potential sources of bias associated with selecting controls from veterinary registered populations of cats due to differences between registered and unregistered cats. Cat owners reported that 13.6% (84/616) of their cats had not been registered with a veterinary practice since living at their current address. Multivariable logistic regression indicated that unregistered cats were significantly more likely than registered cats to be entire, to have not been vaccinated within the previous year, to be living in households in Northern Ireland and in households with an annual income <£10,000.(1) Whilst the neuter status and the vaccination status of the cat are likely to result from non-registration, the household location and annual income are factors that can be used to inform future interventions designed to increase the proportion of veterinary registered cats.

First WNK4-hypokalemia animal model identified by genome-wide association in Burmese cats.

Burmese is an old and popular cat breed, however, several health concerns, such as hypokalemia and a craniofacial defect, are prevalent, endangering the general health of the breed. Hypokalemia, a subnormal serum potassium ion concentration ([K(+)]), most often occurs as a secondary problem but can occur as a primary problem, such as hypokalaemic periodic paralysis in humans, and as feline hypokalaemic periodic polymyopathy primarily in Burmese. The most characteristic clinical sign of hypokalemia in Burmese is a skeletal muscle weakness that is frequently episodic in nature, either generalized, or sometimes localized to the cervical and thoracic limb girdle muscles. Burmese hypokalemia is suspected to be a single locus autosomal recessive trait. A genome wide case-control study using the illumina Infinium Feline 63K iSelect DNA array was performed using 35 cases and 25 controls from the Burmese breed that identified a locus on chromosome E1 associated with hypokalemia. Within approximately 1.2 Mb of the highest associated SNP, two candidate genes were identified, KCNH4 and WNK4. Direct sequencing of the genes revealed a nonsense mutation, producing a premature stop codon within WNK4 (c.2899C>T), leading to a truncated protein that lacks the C-terminal coiled-coil domain and the highly conserved Akt1/SGK phosphorylation site. All cases were homozygous for the mutation. Although the exact mechanism causing hypokalemia has not been determined, extrapolation from the homologous human and mouse genes suggests the mechanism may involve a potassium-losing nephropathy. A genetic test to screen for the genetic defect within the active breeding population has been developed, which should lead to eradication of the mutation and improved general health within the breed. Moreover, the identified mutation may help clarify the role of the protein in K⁺ regulation and the cat represents the first animal model for WNK4-associated hypokalemia.

Antigen specificity of the humoral immune response to Mycoplasma haemofelis infection.

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 crude M. haemofelis antigen 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 with M. 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 the M. haemofelis antigen preparation. The M. haemofelis antigen 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 the M. haemofelis antigen 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 of M. haemofelis antigens 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.

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears.

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.