Commercially Available Enzyme-Linked Immunosorbent Assay and Polymerase Chain Reaction Tests for Detection of Feline Immunodeficiency Virus Infection.

BACKGROUND: Feline immunodeficiency virus (FIV) infection is an important cause of disease of cats worldwide. Initial screening is commonly performed by commercially available point-of-care (POC) ELISA tests. Confirmatory testing for positive POC test results is recommended. Polymerase chain reaction (PCR) tests for FIV are commonly used additional testing methods; however, reported measures of diagnostic accuracy vary widely between PCR tests, making interpretation of results difficult. HYPOTHESIS/OBJECTIVE: There is very good agreement between results of a commercially available PCR test and a POC ELISA test for FIV for specimens collected from owned and shelter-housed cats. ANIMALS: Blood samples from 168 cats from 2 adoption guarantee shelters, an FIV Sanctuary, and 64 private homes were used. METHODS: This was a prospective study. Whole blood samples were collected in K2 -EDTA, divided, and submitted for PCR and ELISA testing. Follow-up whole blood samples were collected in lithium heparin from cats with discordant results and submitted for virus isolation (VI). RESULTS: There was very good agreement between ELISA and PCR (kappa 0.87; P < .001; 95% CI 0.79, 0.95). Of 168 cats, eleven had discordant ELISA/PCR results: 7 ELISA+/PCR- and 4 ELISA-/PCR+. Using VI as a reference standard, there were 4 false-positive PCR results, 5 false-positive ELISA results, and 1 false-negative PCR result (1 cat lost to follow-up). CONCLUSIONS AND CLINICAL IMPORTANCE: While there was good agreement between the POC ELISA and PCR tests, the discordant results highlight the importance of cautious interpretation of test results and the necessity of confirmatory testing.

Exposure to raccoon polyomavirus (RacPyV) in free-ranging North American raccoons (Procyon lotor).

There is evidence that raccoon polyomavirus is causative for neuroglial brain tumors in the western United States. It is unknown if infection is limited to geographic locales where tumors have been reported or is widespread, like human polyomaviruses. We demonstrate raccoons in western, eastern and midwestern states have been exposed to RacPyV by detection of antibodies to capsid protein, VP1. While raccoons in eastern and midwestern states are seropositive, exposure is lower than in the western states. Additionally, across geographic areas seropositivity is higher in older as compared to younger raccoons, similar to polyomavirus exposure in humans. Serum titers are significantly higher in raccoons with tumors compared to raccoons without. Unlike polyomavirus-associated diseases in humans, we did not detect significant sequence variation between tumor and non-tumor tissue in raccoons with tumors compared to those without tumors. This warrants further investigation into co-morbid diseases or genetic susceptibility studies of the host.

Equine coronavirus: An emerging enteric virus of adult horses.

Equine coronavirus (ECoV) is an emerging virus associated clinically and epidemiologically with fever, depression, anorexia and less frequently colic and diarrhoea in adult horses. Sporadic cases and outbreaks have been reported with increased frequency since 2010 from Japan, the USA and more recently from Europe. A faeco-oral transmission route is suspected and clinical or asymptomatic infected horses appear to be responsible for direct and indirect transmission of ECoV. A presumptive clinical diagnosis of ECoV infection may be suggested by clinical presentation, haematological abnormalities such as leucopenia due to lymphopenia and/or neutropenia. Confirmation of ECoV infection is provided by specific ECoV nucleic acid detection in faeces by quantitative PCR (qPCR) or demonstration of coronavirus antigen by immunohistochemistry or electron microscopy in intestinal biopsy material obtained ante or post mortem. The disease is generally self-limiting and horses typically recover with symptomatic supportive care. Complications associated with disruption of the gastrointestinal barrier have been reported in some infected horses and include endotoxaemia, septicaemia and hyperammonaemia-associated encephalopathy. Although specific immunoprophylactic measures have been shown to be effective in disease prevention for closely-related coronaviruses such as bovine coronavirus (BCoV), such strategies have yet not been investigated for horses and disease prevention is limited to basic biosecurity protocols. This article reviews current knowledge concerning the aetiology, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

Infectious diseases in large-scale cat hoarding investigations.

Animal hoarders accumulate animals in over-crowded conditions without adequate nutrition, sanitation, and veterinary care. As a result, animals rescued from hoarding frequently have a variety of medical conditions including respiratory infections, gastrointestinal disease, parasitism, malnutrition, and other evidence of neglect. The purpose of this study was to characterize the infectious diseases carried by clinically affected cats and to determine the prevalence of retroviral infections among cats in large-scale cat hoarding investigations. Records were reviewed retrospectively from four large-scale seizures of cats from failed sanctuaries from November 2009 through March 2012. The number of cats seized in each case ranged from 387 to 697. Cats were screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) in all four cases and for dermatophytosis in one case. A subset of cats exhibiting signs of upper respiratory disease or diarrhea had been tested for infections by PCR and fecal flotation for treatment planning. Mycoplasma felis (78%), calicivirus (78%), and Streptococcus equi subspecies zooepidemicus (55%) were the most common respiratory infections. Feline enteric coronavirus (88%), Giardia (56%), Clostridium perfringens (49%), and Tritrichomonas foetus (39%) were most common in cats with diarrhea. The seroprevalence of FeLV and FIV were 8% and 8%, respectively. In the one case in which cats with lesions suspicious for dermatophytosis were cultured for Microsporum canis, 69/76 lesional cats were culture-positive; of these, half were believed to be truly infected and half were believed to be fomite carriers. Cats from large-scale hoarding cases had high risk for enteric and respiratory infections, retroviruses, and dermatophytosis. Case responders should be prepared for mass treatment of infectious diseases and should implement protocols to prevent transmission of feline or zoonotic infections during the emergency response and when transferring the rescued cats to other shelters or to adopters.

Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

Vascular endothelial growth factor mRNA expression and peritumoral edema in canine primary central nervous system tumors.

Vascular endothelial growth factor (VEGF) is an important regulator of tumor angiogenesis and vascular permeability, and has been implicated both in progression of central nervous system (CNS) tumors and development of vasogenic peritumoral edema. A retrospective study was done to characterize the levels of expression of the 3 major canine VEGF isoforms (VEGF(120), VEGF(164), VEGF(188)) in a variety of spontaneous canine CNS tumors using quantitative TaqMan reverse transcription real-time polymerase chain reaction. Presence and degree of peritumoral edema also were determined in sampled tumors using magnetic resonance imaging (MRI). Increased expression of VEGF relative to normal cerebral cortex tissue was seen predominantly in high grade astrocytic (grade IV) and oligodendroglial (grade III) tumors, with lower expression in low grade astrocytomas (grade II) and meningiomas (grade I). All 3 major VEGF isoforms were present; VEGF(164) was the predominant isoform, particularly in the tumors with the highest VEGF expression. Peritumoral edema was present in all tumor types; however, a significant association between the extent of peritumoral edema and the level of VEGF expression was not apparent.

Molecular quantitative analysis of human viruses in California stormwater.

Many human pathogenic viruses are transmitted via the oral-fecal route and water is one possible vector, representing a risk for public health. Sixty-one large-volume water samples from storm drains in California were processed by a two-step hollow fiber ultrafiltration procedure followed by molecular analysis for human enterovirus and adenovirus types. Each sample was spiked with a surrogate, the benign bacteriophage PP7. Both surrogate and human viruses were quantified by newly designed TaqMan PCR assays. Equations were developed that account for the main variables in the procedure: recovery of the ultrafiltration, efficiency of nucleic acid extraction, and effect of inhibitors on the amplification of viral targets. Adenovirus 40/41 was detected in one sample at 230 genomes per liter, and no other adenovirus or enterovirus types were found. Samples that resulted in nondetects are reported together with the corresponding sample-specific limit of detection (S(LOD)), a useful tool when estimating the public health risk associated with the contact or ingestion of water. Virus concentrations did not correlate with traditional viable indicator concentrations or any of the physicochemical parameters measured. In contrast, coliform concentrations were correlated with total suspended solids. To our knowledge, this is the first study where all factors known to influence limits of detection have been investigated and integrated into equations that are widely applicable to the quantification of viruses or other microbial targets by PCR.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRalpha and c-Met in canine primary brain tumours.

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real-time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]-1, VEGFR-2, endothelial growth factor receptor [EGFR]-1, platelet-derived growth factor receptor a [PDGFRa], and c-Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR-1 and VEGFR-2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR-1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.

Inflammation and changes in cytokine levels in neurological feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a progressive, fatal, predominantly Arthus-type immune-mediated disease that is triggered when cats are infected with a mutant enteric coronavirus. The disease presents variably with multiple organ failure, seizures, generalized effusion, or shock. Neurological FIP is clinically and pathologically more homogeneous than systemic 'wet' or 'dry' FIP; thus, comparison of cytokine profiles from cats with neurological FIP, wet FIP, and non-FIP neurological disease may provide insight into some baseline characteristics relating to the immunopathogenesis of neurological FIP. This study characterizes inflammation and changes in cytokines in the brain tissue of FIP-affected cats. Cellular infiltrates in cats with FIP included lymphocytes, plasma cells, neutrophils, macrophages, and eosinophils. IL-1 beta, IL-6, IL-12, IL-18, TNF-alpha, macrophage inhibitory protein (MIP)-1 alpha, and RANTES showed no upregulation in the brains of control cats, moderate upregulation in neurological FIP cats, and very high upregulation in generalized FIP cats. Transcription of IFN-gamma appeared upregulated in cats with systemic FIP and slightly downregulated in neurological FIP. In most cytokines tested, variance was extremely high in generalized FIP and much less in neurological FIP. Principal components analysis was performed in order to find the least number of 'components' that would summarize the cytokine profiles in cats with neurological FIP. A large component of the variance (91.7%) was accounted for by levels of IL-6, MIP-1 alpha, and RANTES. These findings provide new insight into the immunopathogenesis of FIP and suggest targets for immune therapy of this disease.

The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.

TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.

HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6.

The most common viral disease of cats worldwide is the infection with feline herpesvirus 1 (FeHV-1). This infection may be followed by Herpetic stromal keratitis (HSK), which is supposed to have an immunopathological basis. Experiments using herpes simplex viruses (HSV) in mouse models indicated that HSK may be treated by topical application of the interleukin 10 (IL-10) gene. The objective of this study was the construction of human herpes simplex virus type 1 (HSV-1)-based amplicon vectors expressing feline interleukin genes and delivery of these genes into cells of feline origin. HSV-1-based amplicon vectors encoding either the enhanced green fluorescent protein, the feline IL-6 or the feline IL-10 under control of the HSV-1 immediate-early 4/5 promotor were constructed, packaged into amplicon particles, transduced into feline cells, and tested for RNA synthesis and biological activity. Feline cells were successfully transduced by HSV-1-based amplicon particles and RNA specific for the transgene was detected already at 2h post transduction, with a maximum at 24h. The recombinant feline IL-10 was functionally active as demonstrated by the reduction of both IL-12 p40 and interferon-gamma-mRNA production in Pansorbin stimulated feline peripheral mononuclear cells. Similarly, the recombinant feline IL-6, which was secreted into the supernatant of transduced cells, was able to support the growth of the IL-6-dependent murine B cell hybridoma 7TD1. HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6. It will be of high interest to study the effects of these tools in vivo.

Cytokine mRNA levels in isolated feline monocytes.

Real-time PCR systems were developed to quantitate cytokine expression in short-time cultivated feline monocytes. Feline-specific interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) primers as well as TaqMan probes were designed and were adapted to a quantitative PCR system which had been previously established for feline IL-10 and IL-12 p40. Quantitative analysis of cytokine messenger RNA (mRNA) transcription based on the comparison of the cytokine with the housekeeping gene feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing universally expressed mRNA. GAPDH mRNA was readily detectable in cDNA prepared from short-time cultivated peripheral blood monocytes. Cytokine mRNA was demonstrated in all samples at variable amounts. IL-1beta and TNF-alpha mRNA was constitutively expressed whereas IL-6, IL-10 and IL-12 p40 mRNA was generally expressed at a lower level and was occasionally not detected. There was a great variability of cytokine production between individual cats and at different time points in the same cat.

Upregulation of mRNA of interleukin-1 and -6 in subchondral cystic lesions of four horses.

This study investigated the potential association of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in subchondral cystic lesions (SCL) in horses. With the technique of in situ hybridisation in paraffin sections of fibrous tissue of SCL and quantitative real-time PCR in fresh frozen fibrous tissue and undecalcified bone sections of SCL embedded in acrylic resin, upregulation of mRNA of both cytokines could be demonstrated. mRNA of IL-1beta was upregulated at the periphery of the cystic lesion adjacent to normal bone, whereas IL-6 mRNA was upregulated within the fibrous tissue found within the centre of the SCL. It was concluded that both cytokines are associated in pathological bone resorption observed in SCL and, in combination with increased production of prostaglandin E2, may be responsible for the slow healing, maintenance or further expansion of the cystic lesions.

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA.

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Modulation of early growth response (EGR) transcription factor-dependent gene expression by using recombinant adenovirus.

Early growth response (EGR) transcription factors link initial cytoplasmic events to long-term alterations of cellular gene expression and are induced by various stimuli. To test their roles in cell physiology, we constructed adenoviral recombinants encoding NGFI-A binding protein 2 (NAB2, a repressor of EGR1, EGR2, and EGR3), EGR1, NAB-insensitive EGR1(I293F) (EGR1*), EGR2, and the NAB-binding, repressive domain 1 (R1) of EGR1. These viruses regulated EGR-dependent expression of GFP and luciferase reporter genes in heterologous expression assays. Infection of a myoblast cell line with EGR1 and EGR1* adenovirus induced the endogenous gene for platelet-derived growth factor A chain (PDGF-A). In addition, in neuroblastoma cells, the two novel EGR1 target genes EGR3 and NAB2 were identified by using adenoviral transfer of EGR1 and EGR1*. Our results demonstrate that recombinant adenovirus is useful to regulate heterologous and endogenous EGR target gene expression, and suggest that EGR transcription factors can autoregulate themselves.

Fibrous tissue of subchondral cystic lesions in horses produce local mediators and neutral metalloproteinases and cause bone resorption in vitro.

OBJECTIVES: To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro. STUDY DESIGN: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed. NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro. ANIMALS: Experiment 1: 32 horses with SCL (n = 8), normal joints (7), and joints with moderate (7) and severe (10) osteoarthritis (OA). Experiment 2: 22 horses with SCL (n = 3), normal joints (7), and chip fractures (12). Experiment 3: Conditioned media of fibrous tissue from 3 horses with SCL of the medial femoral condyle (n = 1), distal metacarpal bone (1), and tarsal bone (1). METHODS: Determinations of local mediator concentrations were made with the Griess assay for NO and an enzyme immunoassay kit for PGE2 concentrations in biological fluids. Enzyme activities were assessed with radiolabeled substrates indicating collagenolytic, gelatinolytic, and caseinolytic activities. The resorption pit assay was used to assess osteoclast recruitment and activity. RESULTS: Fibrous tissue of SCL produced NO, PGE2, and NMPs. Of all the variables measured, PGE2 concentrations were the highest in cystic tissue of SCL compared with synovial membrane and articular cartilage from normal joints and joints with chip fractures, indicating that this mediator may play an important role in pathological bone resorption associated with SCL. These findings were supported by the observation that conditioned media of SCL tissue were capable of recruiting osteoclasts and increasing their activity. CONCLUSION: Fibrous tissue of SCL released NO, PGE2, and NMPs into the culture media. It is suspected that intralesional fibrous tissue may play an active role in the pathological process of bone resorption occurring in SCL in horses and may be partly responsible for the maintenance, slow healing rate, and expansion of these lesions. CLINICAL RELEVANCE: Understanding the pathogenesis of SCL will help to establish successful therapy in horses affected with SCL.

Viral infections in free-living populations of the European wildcat.

While the importance of viral infections is well studied in domestic cats, only limited information is available on their occurence and prevalence in the European wildcat (Felis silvestris silvestris). The aim of this study was to determine the prevalence of antibodies to feline coronavirus (FCoV), calicivirus (FCV), herpesvirus (FHV), parvovirus (FPV), immunodeficiency virus (FIV), leukemia virus (FeLV), and FeLV antigenemia in 51 European wildcat sera. Samples were collected between 1996 and 1997 from wildcat populations in France, Switzerland, and Germany. Antibodies to FCoV were detected in two cats (4%) and FCoV RNA was detected in feces of one of these two cats. Antibodies to FCV, FHV and FPV were found at relatively low frequencies of 16%, 4%, and 2%, respectively. Antibodies to FIV were not detected. Although antigen and antibodies to FeLV were detected in 49%, and 75%, respectively, no evidence of FeLV-associated pathology was found. From the low prevalence of FCoV, FCV, FHV and FPV infections and from the fact that the European wildcats live solitarily, it was concluded that these viral infections do not spread readily within a population. Therefore, it may be assumed that release into the wild of European wildcats bred in captivity would not bring about a high risk of introducing of these viral infections to the free-ranging wildcats. As an exception, wildcats should be tested for absence of FIV infection before release if they were at risk to acquire this infection from domestic cats.

The role of polymerase chain reaction and its newer developments in feline medicine.

We give a brief overview on the principles of the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative competitive PCR and real-time PCR (TaqMan technology). The literature dealing with PCR and its role in the diagnosis, pathogenesis and research of infectious diseases of the domestic cat is reviewed. Cross-contaminations which occasionally occur during handling of amplified DNA may be an important problem in the PCR laboratory. In many infectious diseases, PCR results are difficult to interpret as their predictive positive and negative values are not always known. Newer assays, such as TaqMan procedures, are becoming increasingly reliable and cost-effective. It can be expected that additional knowledge on how to interpret PCR results will soon be available.