Is feline foamy virus really apathogenic?

Feline foamy virus (FFV) is a retrovirus commonly found in cats. It is generally thought to be apathogenic, making it a suitable candidate as a gene therapy vector. However, there have been reports of association of FFV with chronic progressive arthritis and a cofactor effect with feline immunodeficiency virus. This study investigated experimental FFV infection and whether this was associated with signs of disease. Eight young specific pathogen free cats were inoculated intramuscularly with FFV. The cats were examined twice weekly and blood and pharyngeal samples were taken. Haematology, biochemistry and FFV quantitative polymerase chain reaction (qPCR) were performed. Tissue samples were also collected throughout the six month period. FFV was initially detected by qPCR in the blood within the first two weeks of infection and viraemia persisted throughout the study. Two peaks of viraemia were observed, at day 20 (80-170FFU/ml blood) and day 155 (332-415FFU/ml blood). FFV was also consistently detected in oropharyngeal samples after day 36. Anti-FFV IgG was detected in all cats by ELISA; antibody levels had an early peak around day 35 and then increased again following the second rise in circulating viral load. All cats remained clinically normal, except for one cat with an unrelated gingivitis. None of the cats developed pyrexia. The biochemical profile and blood cell counts remained within normal limits except for one cat with a persistent eosinophilia. Initial fluctuations in white cell counts settled within three weeks and did not deviate outside of the normal ranges. All tissue samples contained FFV DNA; lymphoreticular tissues, salivary gland and lung had the highest viral loads. Although there were no gross pathological lesions on post mortem examination, histologically a mild glomerulonephritis and a moderate interstitial pneumonia were observed in all cats. We conclude that during the six month period of infection, although cats appeared clinically normal, histopathological changes were observed in the lungs and kidneys. Further investigation of the significance of these changes is warranted before FFV is developed as a vector for gene delivery.

Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis and Bordetella bronchiseptica in cats: experience from 218 European catteries.

A full history of the management practices and the prevalence of upper respiratory tract disease (URTD) at 218 rescue shelters, breeding establishments and private households with five or more cats was recorded. Oropharyngeal and conjunctival swabs and blood samples were taken from 1748 cats. The prevalences of feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydophila felis and Bordetella bronchiseptica were determined by PCR on swab samples. An ELISA was applied to determine the prevalence of antibodies to B. bronchiseptica. The rates of detection by PCR of each pathogen in the cats in catteries with and without ongoing URTD were, respectively, FHV 16 per cent and 8 per cent; FCV 47 per cent and 29 per cent; C. felis 10 per cent and 3 per cent; and B. bronchiseptica 5 per cent and 1.3 per cent; the seroprevalences of B. bronchiseptica were 61 per cent and 41 per cent, respectively. There was evidence that FHV, FCV and B. bronchiseptica played a role in URTD. The risk factors associated with the disease were less than excellent hygiene, contact with dogs with URTD, and larger numbers of cats in the cattery or household.

Transfer of spinal cord material to subsequent bovine carcasses at splitting.

During the slaughter process, cattle carcasses are split by sawing centrally down the vertebral column, resulting in contamination of each half with spinal cord material. Using a novel method based on a real-time PCR assay, we measured saw-mediated tissue transfer among carcasses. Up to 2.5% of the tissue recovered from each of the five subsequent carcasses by swabbing the split vertebral face came from the first carcass to be split; approximately 9 mg was spinal cord tissue. Under controlled conditions in an experimental abattoir, between 23 and 135 g of tissue accumulated in the saw after splitting five to eight carcasses. Of the total tissue recovered, between 10 and 15% originated from the first carcass, and between 7 and 61 mg was spinal cord tissue from the first carcass. At commercial plants in the United Kingdom, between 6 and 101 g of tissue was recovered from the saw, depending on the particular saw-washing procedure and number of carcasses processed. Therefore, if a carcass infected with bovine spongiform encephalopathy were to enter the slaughter line, the main risk of subsequent carcass contamination would come from the tissue debris that accumulates in the splitting saw. This work highlights the importance of effective saw cleaning and indicates that design modifications are required to minimize the accumulation of spinal cord tissue debris and, hence, the risk of cross-contamination of carcasses.

FIP: a novel approach to vaccination. Proceedings from the 2nd International FCoV/FIP Symposium, Glasgow, 4-7 August 2002.

Feline infectious peritonitis (FIP) is a fatal disease of cats. Early attempts at vaccination have been unsuccessful, some even serving to exacerbate the disease through antibody-dependent enhancement. Replication-incompetent feline foamy virus (FFV) transducing vectors are being developed as potential vaccine agents, into which immunogenic fragments of feline coronavirus (FCoV) proteins will be inserted. To use a recombinant viral vector to express FCoV proteins, the agent chosen should be apathogenic and replication incompetent within the host following gene delivery. Spumaviruses confer several advantages over the more traditionally explored retroviral vectors. Stable helper cell line clones have been established by transfection of CRFK cells with FFV tas and assessed using beta-galactosidase assays, PCR, immunofluorescence and western blotting. The generation of infectious virions using these cell lines has been investigated using tas-deleted FFV vectors containing the enhanced green fluorescent protein (eGFP) cassette.

Efficacy of azithromycin for the treatment of feline chlamydophilosis.

The current recommended treatment for feline chlamydophilosis involves daily oral administration of antimicrobials to all cats within an affected group for a prolonged period of time (4-6 weeks). Not surprisingly, owner compliance can be poor resulting in apparent treatment failure. Recent anecdotal evidence, supported by its efficacy in the treatment of Chlamydia trachomatis infection in humans, has suggested that azithromycin may offer an alternative by allowing less frequent dosing for a shorter duration. A clinical trial was designed to evaluate the efficacy of azithromycin for the treatment of chlamydia (Chlamydophila felis) infection in cats. Whilst azithromycin, given at 10-15 mg/kg daily for 3 days and then twice weekly, provided a similar, rapid resolution of clinical signs and negative isolation scores as doxycycline, C felis was re-isolated in four out of the five cats treated. Furthermore, even daily administration of azithromycin to chronically infected cats was ineffective in clearing infection. The azithromycin protocols used here were therefore found to be unsuccessful in eliminating the carriage of this strain of C felis.

Phylogenetic analysis of hemoplasma species: an international study.

Nearly complete 16S rRNA gene sequences for feline and canine hemoplasma isolates from Europe, Australia, Africa, and Asia showed almost 100% identity to those previously reported for United States isolates. Partial sequences of the RNA subunit of the RNase P gene were also determined, and RNase P-based phylogenetic analysis showed that the hemoplasmas are most closely related to the members of the Mycoplasma pneumoniae group.

Use of a PCR assay to assess the prevalence and risk factors for Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in cats in the United Kingdom.

Blood samples from 426 healthy and sick cats in the UK were tested in a PCR assay for 'Candidatus Mycoplasma haemominutum' and Mycoplasma haemofelis (basonym Haemobartonella felis). Seventy-two of the cats (16.9 per cent) were positive for 'Candidatus M. haemominutum' alone, six (1.4 per cent) were positive for M. haemofelis alone and one (0.2 per cent) was positive for both. Logistic regression analysis indicated that older male cats were significantly more likely to be infected with 'Candidatus M. haemominutum', but there was no significant association between it and any of the haematological variables measured. M. haemofelis infection was uncommon in the anaemic cats sampled, and there were too few positive cases for multivariable analysis to be performed for M. haemofelis-positive status.

Protection against oronasal challenge with virulent feline leukaemia virus lasts for at least 12 months following a primary course of immunisation with Leukocell 2 vaccine.

The duration of immunity provided by a feline leukemia virus (FeLV) vaccine, Leukocell 2, was determined. Kittens were vaccinated when 9 and 12 weeks of age and were challenged 12 months later with FeLV-A/Glasgow-1. An oronasal challenge protocol without corticosteroid enhancement was developed in order to induce a persistent viraemia in a high proportion of adult cats. Fourteen of 18 (80%) of the vaccinated cats challenged in this way remained non-viraemic while 9/15 (60%) of age-matched controls became persistently infected, a preventable fraction of 63%. This difference was statistically significant (P=0.038). For comparison, 10 of 12 (83%) 15-17-week-old kittens challenged in the same way became persistently infected, confirming the relative resistance of adult animals to FeLV. Tests for virus neutralising and anti-feline oncornavirus-associated cell membrane antigen (FOCMA) antibodies suggested that the former were more important than the latter in protection. Thus, Leukocell 2 protected a significant proportion of cats from FeLV challenge 1 year after primary vaccination as kittens.

Detection of antibodies to Borna disease virus (BDV) in Turkish horse sera using recombinant p40. Brief report.

The nucleoprotein of Borna disease virus (BDV-p40) was produced in a Baculovirus expression system using sf9 cells. The purity and specificity of the recombinant p40 was confirmed by SDS-PAGE and immunoblotting. The recombinant p40 was used in an ELISA to screen horse sera in Turkey. For this, 323 horses from selected cities in the Marmara region of Turkey were examined clinically and serum was collected from each. All horses were clinically healthy except for a few with wounds on the skin. Antibodies to BDV were detected in the sera of 82 (25%) of 323 horse sera. Six sera were selected that had low, medium or high OD values by ELISA and were analysed by Western blotting. All reacted specifically with p40 at a dilution of 1 in 1000. This is the first report of the detection of Borna disease in Turkey and needs further molecular biological investigations to compare the Turkish strains with those strains detected in Europe.

Targeted lymph node immunization can protect cats from a mucosal challenge with feline immunodeficiency virus.

With the rapid spread of human immunodeficiency virus (HIV) infection worldwide it is clear that effective strategies for mucosal vaccination against lentiviruses are urgently required. The aim of the present study is to determine whether protective immune responses against a mucosal challenge by feline immunodeficiency virus (FIV) can be elicited by targeting the immunization to the medial iliac lymph nodes--the principal site of migration of cells from the genital and rectal mucosa. Cats were challenged with homologous FIV via the rectal route. Targeted lymph node immunization was found to be an effective route of immunization eliciting both humoral and proliferative responses to peptide-based and fixed cell vaccines. Vaccination with fixed virus infected cells elicited protection against a cell-free mucosal FIV challenge. In addition, some cats vaccinated with fixed uninfected cells also remained uninfected following a cell-associated FIV challenge.

Controlled study of the efficacy of clavulanic acid-potentiated amoxycillin in the treatment of Chlamydia psittaci in cats.

Twenty-four specific pathogen-free cats were inoculated with 3 x 10(3) infectious units of a field isolate of Chlamydia psittaci on to the corneal surface. Seven days later they were assigned randomly to three groups of eight and treated orally for 19 days with either clavulanic acid-potentiated amoxycillin, doxycycline or a placebo. Both treated groups responded rapidly, with a marked reduction in isolation rates and clinical scores which were significantly lower than in the placebo group within two and four days, respectively. After two days the group treated with potentiated amoxycillin had a significantly lower isolation score than the group treated with doxycycline. Forty days after they were infected the clinical signs recurred in five of the eight cats treated with potentiated amoxycillin, but a four-week course of potentiated amoxycillin resulted in a complete clinical recovery with no evidence of a recurrence for six months.

16S rDNA comparison demonstrates near identity between an United Kingdom Haemobartonella felis strain and the American California strain.

A handful of North American (USA) strains of the uncultured erythrocytotrophic pathogen of cats, Haemobartonella felis, have been differentiated by comparison of the 16S rRNA gene sequences. Using this approach, an UK strain was characterised, providing an identity for a non-USA H. felis for the first time. This strain shared close phylogenetic homology with the USA Californian strain.

Detection of antibodies to Borna disease virus in Turkish cats by using recombinant p40.

Recombinant p40 produced by baculovirus was used in an ELISA to screen samples of serum taken from 80 cats in Istanbul. The sera were also analysed for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Antibodies to Borna disease virus- (BDV) p40 were detected in 34 (42-5 per cent) of the 80 cats. Seventy-three per cent of the sera which were positive for FIV and 26 per cent of the sera which were negative for FIV had antibodies to BDV. There was no difference in the percentage of sera which were positive for BDV between the cats that were positive or negative for FeLV. Three of the cats had neurological disease and two of these had antibodies to BDV. Six sera with low, medium or high optical densities (ODS) by ELISA were analysed by Western blotting. Only the sera with medium and high ODS reacted specifically with p40 at a dilution of 1 in 1,000.

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat (Felis domesticus) Ig epsilon constant region.

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.

Demonstration of thiopurine methyltransferase activity in the erythrocytes of cats.

Azathioprine is a purine analogue used as an immunosuppressive and immunomodulator agent in various mammals, including cats. Several adverse reactions have been reported and have limited the use of the drug in the cat. Adverse reactions to azathioprine in humans have been correlated with reduced activity of thiopurine methyltransferase (TPMT) in erythrocytes. The purpose of this preliminary study was to determine if cats have TPMT activity in their erythrocytes and to compare the values obtained with the normal range for humans and the normal range for dogs in a preliminary report. Activity of the enzyme was measured in blood samples drawn from 41 cats. Blood also was taken from 5 dogs. The mean erythrocyte TPMT activity in the cats was 2.4 +/- 0.4 nmol (range, 1.2-3.9 nmol) per hour per milliliter of red blood cells (U/mL RBC) or 2-8 nmol per hour per gram of hemoglobin (U/g Hb). This range was far lower than the normal human range (8-15 U/mL RBC; 16-33 U/g Hb) and was of monopolar distribution. This observation apparently precludes any diagnostic purpose in assaying erythrocyte TPMT in this species. Erythrocyte TPMT activity in the 5 dogs ranged from 5.5 to 13.1 U/mL RBC (11-27 U/g Hb), which was comparable with normal and carrier ranges for humans, but proof of TPMT genetic polymorphism in either species will require genotyping studies.

Methods for detection of haematogenous dissemination of brain tissue after stunning of cattle with captive bolt guns.

Because of concern that the stunning of cattle with captive bolt guns (CBGs) could, if used on an animal with bovine spongiform encephalopathy (BSE), cause embolism of infective brain tissue and carcass contamination, the Ministry of Agriculture, Food and Fisheries commissioned research to assess the risk of haematogenous dissemination of CNS material after stunning. We have devised two methods to investigate this risk. The first involves the concentration of embolic tissue in buffy coat Cytoblocks that can be embedded for sectioning, microscopy and immunocytochemistry. The second method is an ELISA for the presynaptic protein, syntaxin 1B. The methods were validated by analysis of several bovine tissues, including blood samples deliberately contaminated with brain. We then studied jugular venous blood obtained before and after the stunning of 60 cattle with CBGs. Samples obtained, after stunning, from five of the cattle contained CNS tissue within the Cytoblocks and yielded positive syntaxin assays. Syntaxin was also detected in samples from one other animal that had been stunned with a pneumatically operated CBG. The described methods should allow an assessment of the risk of neuroembolism associated with different types of CBG and may also be useful in other contexts.

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge.

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund's adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

PCR-based 16S ribosomal DNA detection technique for Clostridium estertheticum causing spoilage in vacuum-packed chill-stored beef.

Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia. Clostridium estertheticum was first isolated from vacuum-packed beef from southern Africa, but has recently been found in beef originating from northern Europe. This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised for use in measures to control the bacterium in the abattoir and to study its ecology. In the first method, primers were designed having a high annealing temperature of 65 degrees C to increase specificity, producing a PCR product of 567 bp from the 16S rDNA. Two species of Enterobacteriaceae found in meat cross-reacted in this test, and so it was necessary to use a second step, digesting the PCR product with two restriction enzymes. Subsequently a further set of primers was designed, producing a PCR product of 641 bp, and using an annealing temperature of 60 degrees C. The second procedure was more specific and did not require subsequent restriction analysis of the PCR product. The two sets of primers appeared to have similar sensitivity, detecting 10-100 cells of C. estertheticum in broth, meat or meat purge (drip). A semiquantitative method is described for estimating numbers of the target bacterium.