Quantification and molecular characterization of the feline leukemia virus A receptor.

Virus receptors and their expression patterns on the cell surface determine the cell tropism of the virus, host susceptibility and the pathogenesis of the infection. Feline thiamine transport protein 1 (fTHTR1) has been identified as the receptor for feline leukemia virus (FeLV) A. The goal of the present study was to develop a quantitative, TaqMan real-time PCR assay to investigate fTHTR1 mRNA expression in tissues of uninfected and FeLV-infected cats, cats of different ages, in tumor tissues and leukocyte subsets. Moreover, the receptor was molecularly characterized in different feline species. fTHTR1 mRNA expression was detected in all 30 feline tissues investigated, oral mucosa scrapings and blood. Importantly, identification of significant differences in fTHTR1 expression relied on normalization with an appropriate reference gene. The lowest levels were found in the blood, whereas high levels were measured in the oral mucosa, salivary glands and the musculature. In the blood, T lymphocytes showed significantly higher fTHTR1 mRNA expression levels than neutrophil granulocytes. In vitro activation of peripheral blood mononuclear cells with concanavalin A alone or followed by interleukin-2 led to a transient increase of fTHTR1 mRNA expression. In the blood, but not in the examined tissues, FeLV-infected cats tended to have lower fTHTR1 mRNA levels than uninfected cats. The fTHTR1 mRNA levels were not significantly different between tissues with lymphomas and the corresponding non-neoplastic tissues. fTHTR1 was highly conserved among different feline species (Iberian lynx, Asiatic and Indian lion, European wildcat, jaguarundi, domestic cat). In conclusion, while ubiquitous fTHTR1 mRNA expression corresponded to the broad target tissue range of FeLV, particularly high fTHTR1 levels were found at sites of virus entry and shedding. The differential susceptibility of different species to FeLV could not be attributed to variations in the fTHTR1 sequence.

Thoracic computed tomography findings in dogs experimentally infected with Angiostrongylus vasorum.

To characterize the computed tomography (CT) features of thoracic lesions caused by infection with Angiostrongylus vasorum, pre- and postcontrast CT was performed in six experimentally infected Beagles 13 weeks postinoculation and in four of these 9 weeks postchemotherapy. Findings were compared with survey radiographs and necropsy findings. A multicentric bronchoalveolar pattern more pronounced at the lung periphery was present radiographically. On CT, the predominant abnormality underlying this alveolar pattern was multiple large nodules merging to areas of consolidation, and containing air bronchograms of varying extent. These nodular changes corresponded to histopathologic granulomata, consisting mainly of macrophages, multinucleated giant cells, and lymphocytes that had accumulated around larvae and eggs. Morphologically, no bronchial changes were observed on CT or histologically. Quantitatively, however, on CT there was evidence of bronchial thickening at 13 weeks postinoculation and mild very peripheral bronchiectasia 9 weeks postchemotherapy. Regional lymph nodes were enlarged after infection, and smaller after treatment. On postcontrast CT, several suspicious intraluminal filling defects suggestive of thrombosis were found; however, the tortuosity of some pulmonary arteries seen radiographically was not present in CT images. After treatment, the consolidations and large nodules had almost completely disappeared. A remaining radiographic interstitial pattern was characterized on CT as ground-glass opacifications, subpleural interstitial thickening, subpleural lines, and interface signs. These interstitial changes reflected fibrosis as documented histopathologically. CT allowed very detailed and accurate characterization of pulmonary parenchymal lesions, bronchi, and lymphnodes and closely reflected histopathological changes.

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum.

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n=3) or 500 (n=3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47-49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident.

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma.

BACKGROUND: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. RESULTS: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. CONCLUSIONS: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

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

BACKGROUND: Gene 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. RESULTS: RNA 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), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-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. CONCLUSION: The 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.

A cross sectional study of prevalence, risk factors, population attributable fractions and pathology for foot and limb lesions in preweaning piglets on commercial farms in England.

BACKGROUND: In a cross sectional study of 88 indoor and outdoor English pig farms, the prevalence of foot and limb lesions in 2843 preweaning piglets aged 1-4 weeks from 304 litters was recorded. The environmental risks for the prevalence of lesions and population attributable fractions were calculated. The risks for lesions in piglets were compared with those for limb and body lesions in their mothers. A small number of piglets with each type of lesion were examined post mortem to elucidate the pathology of the clinical lesions observed. RESULTS: The prevalence of sole bruising, sole erosion, skin abrasion and swollen joints or claws in 2843 piglets was 49.4% (1404), 15.5% (441), 43.6% (1240) and 4.7% (143) respectively. The prevalence of all foot and limb lesions was higher in indoor housed piglets than in outdoor housed piglets. The prevalence of sole bruising (OR 0.3) and skin abrasion (OR 0.6) decreased with each week of age from 1-4 weeks, but there was no significant association between piglet age and the prevalence of sole erosion or swollen joints and claws. There was an increased prevalence of sole bruising (OR 3.0) and swollen joints or claws (OR 3.0) and a decreased prevalence of skin abrasion (OR 0.3, piglets

Occurrence of cataract and fatty liver in captive plains viscachas (Lagostomus maximus) in relation to diet.

An increased incidence of cataract and fatty liver in plains viscachas (Lagostomus maximus) was noted for many years at the Zurich Zoo (Switzerland). Based on elevated serum fructosamine and glucose, diabetes mellitus was diagnosed; and these parameters normalized when the diet of the animals was changed from a low-fiber to a high-fiber diet. In this present study, 177 necropsy reports from before and after the diet changes were evaluated for the incidence of cataracts and fatty liver. Sixteen of 56 animals (29%) that were born before the diet change developed cataract. In contrast, only two of 121 animals (1.65%) that were born after the diet change developed cataract. The prevalence of cataract and fatty liver in animals born after the diet change (1% and 0%, respectively) was significantly lower than in animals born before (9% and 6%, respectively) the diet change. The results suggest that the plains viscachas at the Zurich Zoo were affected by an alimentary-induced diabetes mellitus (type 2) before the diet was changed.

Vaccination against the feline leukaemia virus: outcome and response categories and long-term follow-up.

Feline leukaemia virus (FeLV) is a pathogen inducing fatal disease in cats worldwide. By applying sensitive molecular assays, efficacious commonly used FeLV vaccines that protect cats from antigenaemia were found not to prevent proviral integration and minimal viral replication after challenge. Nonetheless, vaccines protected cats from FeLV-associated disease and prolonged life expectancy. The spectrum of host response categories was refined by investigating plasma viral RNA loads. All cats initially fought similar virus loads, although subsequently loads were associated with infection outcomes. Persistence of plasma viral RNA was moderately associated with reactivation of FeLV infection. In conclusion, sensitive molecular assays are important tools for reviewing pathogenesis of FeLV infection.

Pansteatitis and severe hypocalcaemia in a cat.

A 13-year-old Siamese cat was presented for investigation of lethargy and progressive abdominal enlargement. Serum chemistry revealed severe reduction of total and ionised serum calcium. The omentum appeared hyperechoic with scattered hypoechoic foci on abdominal ultrasound examination. Elevated serum parathormone and low fractional excretion of calcium excluded a parathyroid disorder and renal loss of the electrolyte. During laparotomy the omentum appeared opaque, white and firm. Post-mortem examination revealed that the thoracic and subcutaneous fat was also affected. Histopathology confirmed the diagnosis of pansteatitis with diffuse calcium soaps formation. While, severe hypocalcaemia is occasionally seen in cats, the association with pansteatitis has not been reported previously. In man, a cause-and-effect relationship between calcium soaps and hypocalcaemia is recognised, though the association is rare.

Haycocknema-like nematodes in muscle fibres of a horse.

A 14-year-old horse (imported to Switzerland from Ireland 8 years earlier) showed signs of chewing muscle atrophy. A severe chronic myositis, caused by numerous immature and mature female nematodes, was diagnosed in muscle samples obtained by biopsy and subsequently at necropsy. Most of the nematodes had invaded muscle fibres of the masseter, root of the tongue and anterior breast, only a few were found in the intermuscular interstitium. Isolated nematodes and parasite sections were clearly different from muscle larvae of Trichinella spp. but showed morphological similarities to Haycocknema perplexum, a nematode species (order Enoplida, family Robertdollfusidae) recently found in the musculature of a human patient in Australia. However, our material did not allow the precise identification of the nematode genus nor the unequivocal differentiation from Halicephalobus gingivalis. This species infects horses and humans and can cause severe granuloma formation in muscles and many other organ systems, but has never been observed to invade individual muscle fibres. Our findings show that nematodes of another genus than Trichinella may invade muscle fibres of the horse and cause myositis. These nematodes are provisionally regarded as Haycocknema-like.

Fine-mapping and candidate gene analysis of bovine spinal muscular atrophy.

Bovine spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease, has been mapped at moderate resolution to the distal part of Chromosome 24. In this article we confirm this location and fine-map the SMA locus to an interval of approximately 0.8 cM at the very distal end of BTA24. Despite remarkable similarity to human SMA, the causative gene SMN can be excluded in bovine SMA. However, the interval where the disease now has been mapped contains BCL2, like SMN an antiapoptotic factor, and shown to bind to SMN. Moreover, knockout mice lacking the BCL2 gene show rapid motor neuron degeneration with early postnatal onset, as observed in bovine SMA. A comparative cattle/human map of the distal end of BTA24, based on the emerging bovine genome sequencing data, shows conserved synteny to HSA18 with hints of a segmental duplication and pericentric inversion just after the last available bovine marker DIK4971. This synteny lets us conclude that SMA is in immediate vicinity of the telomere. Candidate gene analysis of BCL2, however, excludes most of this gene, except its promoter region, and draws attention to the neighboring gene VPS4B, part of the endosomal protein-sorting machinery ESCRT-III which is involved in several neurodegenerative diseases.

Reassessment of feline leukaemia virus (FeLV) vaccines with novel sensitive molecular assays.

We previously described antigen negative, provirus positive cats. Subsequently, we hypothesized that efficacious FeLV vaccines cannot prevent minimal viral replication. Thus, we vaccinated cats with either a canarypox-vectored live or a killed virus vaccine and analyzed the challenge outcome with quantitative PCR and a newly established real-time RT-PCR. When judged by conventional parameters (antigenaemia, virus isolation), most of the vaccinated cats were, as expected, protected from persistent viraemia. However, all cats were found to be plasma viral RNA positive. The loads were significantly associated with the infection outcome. Thus, commonly used FeLV vaccines understood to be successful model antiretroviral vaccines protecting against FeLV-related diseases do not confer sterilizing immunity.

Transgenic mice overexpressing erythropoietin adapt to excessive erythrocytosis by regulating blood viscosity.

Severe elevation of red blood cell number is often associated with hypertension and thromboembolism resulting in severe cardiovascular complications. However, some individuals such as high altitude dwellers cope well with an increased hematocrit level. We analyzed adaptive mechanisms to excessive erythrocytosis in our transgenic (tg) mice that, due to hypoxia-independent erythropoietin (Epo) overexpression, reached hematocrit values of 0.8 to 0.9 without alteration of blood pressure, heart rate, or cardiac output. Extramedullar erythropoiesis occurred in the tg spleen, leading to splenomegaly. Upon splenectomy, hematocrit values in tg mice decreased from 0.89 to 0.62. Tg mice showed doubled reticulocyte counts and an increased mean corpuscular volume. In tg mice, plasma volume was not elevated whereas blood volume was up to 25% of the body weight compared with 8% in wild-type (wt) siblings. Although plasma viscosity did not differ between tg and wt mice, tg whole-blood viscosity increased to a lower degree (4-fold) than expected from corresponding hemoconcentrated wt blood (8-fold). This moderate increase in viscosity is explicable by the up to 3-fold higher elongation of tg erythrocytes at physiologic shear rates. Apart from the nitric oxide-mediated vasodilation we reported earlier, adaptation to high hematocrit levels in tg mice involves regulated elevation of blood viscosity by increasing erythrocyte flexibility.