Barbara Riond

In vivo transmission studies of 'Candidatus Mycoplasma turicensis' in the domestic cat

The natural transmission routes of the three feline haemotropic mycoplasmas – Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’ (CMt) – are largely unknown. Since CMt has been detected in the saliva of infected cats using PCR, we hypothesised that direct transmission via social or aggressive contact may occur. The aim of this study was to evaluate this transmission route. CMt-positive saliva and blood samples were obtained from three prednisolone-treated specific pathogen-free (SPF) cats that were infected intraperitoneally with CMt. Five SPF cats were inoculated with CMt-positive saliva or blood subcutaneously to mimic cat bites, and five cats were inoculated orally with blood or oronasally with saliva to mimic social contact. Blood samples were monitored for CMt infection using quantitative real-time PCR and for seroconversion using a novel western blot assay. Neither oronasal nor subcutaneous inoculation with CMt-positive saliva led to CMt infection in the recipient cats, as determined by PCR, independent of prior prednisolone treatment. However, when blood containing the same CMt dose was given subcutaneously, 4 of the 5 cats became PCR-positive, while none of the 5 cats inoculated orally with up to 500 μL of CMt-positive blood became PCR-positive. Subsequently, the latter cats were successfully subcutaneously infected with blood. All 13 CMt-exposed cats seroconverted. In conclusion, CMt transmission by social contact seems less likely than transmission by aggressive interaction. The latter transmission may occur if the recipient cat is exposed to blood from an infected cat.

Evaluation of the i-STAT portable clinical analyzer in chickens (Gallus gallus).

The i-STAT portable clinical analyzer (PCA) was evaluated for performance in avian species. With the EG7+ cartridge, which provided results for hydrogen ion concentration, oxygen tension, carbon dioxide tension, sodium, potassium, ionized calcium, hematocrit, and various calculated parameters, analytical accuracy and precision were tested by comparing obtained values to those of established traditional blood gas and chemistry analyzers. Demings regression and bias plots were used to compare i-STAT results with those obtained by laboratory professionals using benchtop analyzers. The reliability of the i-STAT PCA with EG7+ cartridges was good, with 0–5.7% system failures in measured values. Regression statistics were good for all blood gas analytes and acceptable for electrolytes and calculated parameters, except for potassium and base excess, for which the regression data or the discrepancy between the methods was too large. The system was reliable and easy to use and had an overall acceptable accuracy in avian species. These features, together with portability and small required blood volumes, make the i-STAT suitable for point-of-care use in critical avian patients, although single values require careful interpretation.

How molecular methods change our views of FeLV infection and vaccination

FeLV was discovered 40 years ago and vaccines have been commercially available for almost two decades. So far, most FeLV pathogenesis and vaccine studies were conducted assaying parameters, such as virus isolation and antigen detection. Accordingly, regressive infection was characterized by transient or undetectable viremia, while persistent viremia is typically observed in cats with progressive infection. Using real-time polymerase chain reaction assays, the spectrum of host response categories to FeLV infection was recently refined by investigating proviral and plasma viral RNA loads. Cats believed to be immune to FeLV infection were found to turn provirus-positive after virus exposure. Moreover, efficacious FeLV vaccines were found unable to prevent provirus-integration and minimal viral replication. Remarkably, no difference was found in initial proviral and plasma viral RNA loads between cats with different infection outcomes. Only subsequently, the infection outcome is associated with FeLV loads. FeLV provirus was found to persist for years; reoccurrence of viremia and disease development was observed in some cats. Thus, aviremic provirus-positive cats are FeLV carriers and, following reactivation, may act as an infection source. However, integrated viral DNA may also be essential for solid protection and long-lasting maintenance of protective immunity. In conclusion, real-time TaqMan PCR and RT-PCR assays are highly sensitive and specific. They yield a more sensitive measure for FeLV exposure than antigen detection, virus isolation or immunofluoresence assays. We recommend the use of real-time PCR assays to identify FeLV exposed cats, particularly in catteries, and investigate obscure clinical cases that may be FeLV-associated. The use of sensitive molecular methods will contribute to a more in-depth understanding of the FeLV pathogenesis.

Quantitative TaqMan ® real-time PCR assays for gene expression normalisation in feline tissues

BackgroundGene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.ResultsRNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.ConclusionThe reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis.

BACKGROUND Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and alpha-, beta-, and gamma-globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. OBJECTIVES The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm-blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. METHODS Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi-automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within-run and within-assay precision. Data from warm-blooded and draught horses were compared using the Mann-Whitney U test. RESULTS Within-run and within-assay CVs were <5% for all protein fractions. No significant difference was found between warm-blooded and heavy draught horses and so combined reference intervals (2.5-97.5%) were calculated for total protein (51.0-72.0 g/L), albumin (29.6-38.5 g/L), alpha(1)-globulin (1.9-3.1 g/L), alpha(2)-globulin (5.3-8.7 g/L), beta(1)-globulin (2.8-7.3g/L), beta(2)-globulin (2.2-6.0 g/L), and gamma-globulin (5.8-12.7 g/L) concentrations, and albumin/globulin ratio (0.93-1.65). CONCLUSION Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.

Evaluation of a novel haematology analyser for use with feline blood

A novel haematology analyser was evaluated for its use with feline samples. Complete blood cell counts, a five-part differential count, and reticulocyte numbers were determined, and the results compared with reference data. Coefficients of correlation, Passing-Bablok regression analysis and Bland-Altmann difference plots with biases and 95% limits of agreement are reported. Precision and linearity were also studied. The instrument demonstrated very low imprecision, and the tested range of linearity exceeded the reference ranges provided by the manufacturer. For all parameters except monocytes (r = 0.65), the analyser results correlated well with reference methods. Compared with the manual count of aggregated reticulocytes, the instrument showed good agreement with a positive bias. The optical platelet count correlated well with the manual chamber count. In conclusion the analyser was found to be highly reliable for the analysis of feline blood samples in a large veterinary laboratory.

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation.

Abstract The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host–virus interactions in felids, we have developed 12 real-time TaqMan® qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM™, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM™ and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.

Hämatologische und klinisch-chemische Referenzwerte für adulte Ziegen und Schafe

Sheep and goat husbandry has always played an important role in swiss agriculture, but in recent years these animals are increasingly appreciated as hobby pets. This opens new diagnostic and therapeutic perspectives for veterinary surgeons, notably in the determination of hematological and clinical biochemical parameters. For the current methods used there are no reference range values available for adult sheep and goats kept under swiss conditions. Therefore, the present study was designed to determine haematological and clinical biochemical parameters in 102 goats and 102 sheep from swiss flocks. Significant differences were found between the two species, especially regarding erythrocyte count, mean erythrocyte volume, hemoglobin content as well as leukocyte count. It is therefore of utmost importance to discriminate between sheep and goats when interpreting laboratory tween findings.

Association between endogenous feline leukemia virus loads and exogenous feline leukemia virus infection in domestic cats

Recently, we demonstrated that endogenous feline leukemia virus (enFeLV) loads may vary among cats of different populations and that FeLV-infected cats have higher enFeLV loads than uninfected cats. Thus, we hypothesized that enFeLV might influence the pathogenesis and outcome of FeLV infection. No significant difference in the infection outcome (regressive versus progressive infection) was observed between groups of cats with high or low enFeLV loads following FeLV-A challenge. However, cats with high enFeLV loads showed higher viral replication (plasma viral RNA and p27 antigen levels) than cats with low enFeLV loads in the early phase of the infection. The enFeLV transcription level varied at different time points, but no clear-cut pattern was observed. In conclusion, our results demonstrated an association between enFeLV loads and FeLV replication but not outcome of infection. enFeLV should be considered as an important confounder in experimental FeLV infection or vaccination studies.