Keratoconjunctivitis in a group of Icelandic horses with suspected γ-herpesvirus involvement.

REASONS FOR PERFORMING STUDY: The role of equid γ-herpesviruses on ocular surface diseases has been disputed, because the diagnosis is usually based on clinical symptoms and detection of viral DNA from samples obtained from live animals. OBJECTIVES: To describe the clinical course, results of polymerase chain reaction (PCR) analysis, in situ hybridisation, cell culture and pathohistological findings of select cases in a presumed outbreak of herpesvirus infection in a group of 15 Icelandic horses. STUDY DESIGN: Case series. METHODS: Pooled ocular and nasal swabs and peripheral blood mononuclear cells of horses diagnosed clinically with herpesvirus-associated keratoconjunctivitis were analysed for presence of equine herpesviruses (EHV)-2 and EHV-5 nucleic acid using real-time PCR. Necropsy specimens from one horse, subjected to euthanasia due to deterioration of clinical symptoms were examined histopathologically, and analysed for presence of EHV-2 and EHV-5 nucleic acid using real-time PCR. In situ hybridisation and cell culture of select samples were performed. RESULTS: All horses with symptoms of severe keratoconjunctivitis were positive for presence of either EHV-2 and/or EHV-5 nucleic acid using real-time PCR. Assessment of necropsy specimens of the most severely affected case, revealed presence of EHV-2 and/or EHV-5 nucleic acid in several ocular and extraocular anatomical locations. The remaining horses responded favourably to symptomatic treatment. CONCLUSIONS: This case series illustrates a severe outbreak of keratoconjunctivitis in a group of Icelandic horses, with suspected γ-herpesvirus involvement. For the first time equid γ-herpesviruses were detected in intraocular anatomical locations.

Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels, Oman, 2013.

A countrywide survey in Oman revealed Middle Eastrespiratory syndrome coronavirus (MERS-CoV) nucleicacid in five of 76 dromedary camels. Camel-derivedMERS-CoV sequences (3,754 nucleotides assembled from partial sequences of the open reading frame (ORF)1a, spike, and ORF4b genes) from Oman and Qatar were slightly different from each other, but closely related to human MERS-CoV sequences from the same geographical areas, suggesting local zoonotic transmission. High viral loads in nasal and conjunctival swabs suggest possible transmission by the respiratory route.

Prevalence of linear keratopathy in a herd of Lipizzaners over an 18-month period.

The prevalence of linear keratopathy with progressing age in a closed population of a single horse breed is reported. All Lipizzaners in three federal states in Austria underwent complete ophthalmic examination four times over a period of 18 months, with six-month intervals. Findings consistent with linear keratopathy were recorded, and associated with factors such as sex, location, boarding system and level of performance throughout the study period. Logistic regression was applied to determine the influence of age on ophthalmic findings. On the first, second, third and fourth examinations, 0.8 per cent, 3.1 per cent, 4.4 per cent and 4.8 per cent (of 266, 261, 249 and 230 horses, respectively) of the study population, were diagnosed with linear keratopathy. This finding was consistently identified in the same horses, and once identified, no further progression was noted. Horses with this finding had no history of previous ocular problems or concurrent ocular abnormalities. Statistical analysis did not reveal any influence of sex, location, boarding, or level of performance; however the prevalence of linear keratopathy was found to increase with progressive age (P<0.5). The results of this study indicate that linear keratopathy was not congenital and was non-progressive in the Lipizzaner over a period of 18 months.

Effect of impregnating agent and relative humidity on surface characteristics of sorbents determined by inverse gas chromatography.

Sorbents that potentially can be used for separation of the products of biotechnological conversion of glycerol were examined. Properties of Zeolite 5A, resins: Amberlite, Diaion and their samples impregnated with an aqueous solutions of 1,2,3-propanetriol, 1,3-propanediol, 2,3-butanediol, acetic acid, succinic acid and model fermentation broth were investigated. Because surface properties will probably depend on the ambient humidity the IGC experiments were carried out under different conditions of relative humidity RH=0, 40 and 80%. Activity of the sorbents surface was expressed by the value of the dispersive component of the free surface energy. Inverse gas chromatography was also used to express acid-base properties of materials described by KA and KD parameters. The changes in the activity of investigated sorbents significantly varied depending on the type of impregnating agent. Moreover, the obtained results demonstrate that humidity can strongly influence, in some cases, the dispersive component of the free surface energy and the ability to specific interactions (KA and KD).

Identification of mixed infections with different genotypes of avian bornaviruses in psittacine birds with proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes.

Natural zoonotic infections of two marmosets and one domestic rabbit with herpes simplex virus type 1 did not reveal a correlation with a certain gG-, gI- or gE genotype.

Infections with herpes simplex virus type 1 (HSV-1) are not restricted to humans but infrequently may be transmitted to certain animal species, in some cases resulting in severe disease, including encephalitis and death. Recent studies demonstrate that humanderived HSV-1 field isolates can be typed according to their gG- gIand gE gene sequences. We investigated whether HSV-1 infections of animals were predominantly caused by a certain genotype. Isolates derived from two marmosets and one domestic rabbit, however, revealed different genotypes. Despite the very limited number of investigated animal-derived HSV-1 strains, this result does not point towards the existence of certain HSV-1 genotypes with a higher potential of being transmitted to animals.

Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species.

Borna disease is a severe viral-induced disorder of the central nervous system of horses, sheep, and a few other animal species, occurring in certain areas of central Europe. Pathogenesis and epidemiology of natural Borna disease virus (BDV) infections are still not fully understood; several unique epidemiologic features, however, point toward the existence of BDV reservoir populations other than the final hosts. In this study, 69 mice and 12 shrews were trapped and examined. The virus distribution was investigated in detail in 2 BDV-positive bicolored white-toothed shrews, Crocidura leucodon, by immunohistochemistry and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). RT-PCR amplification products were sequenced, and the sequences were compared. These shrews had been collected in a BDV-endemic geographical region using live traps and did not show obvious clinical or pathological disease signs. BDV antigen and nucleic acid were identified in several organs, including the brain, mainly in nerve tissue and neurons, respectively, but also in parenchymal cells (eg, hepatocytes, Leydig cells) and epithelial cells, particularly of the respiratory and urogenital tract.

Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Influence of different semen extenders and seminal plasma on PMN migration and on expression of IL-1beta, IL-6, TNF-alpha and COX-2 mRNA in the equine endometrium.

After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oestrous cycle before experiments were started. No differences in volume, pH, specific density or cell count of lavage fluid were found between the treatments. A significantly (p<0.01) lower number of leukocytes in the endometrium was identified in pre-experiment biopsies (68+/-5 leukocytes per field) compared to PBS (154+/-32), SP (175+/-22), SM (193+/-29) and EY treatments (113+/-17). PMN numbers were lower (p<0.01) after infusion of EY (23+/-10) compared to PBS (59+/-21) and SM extender (69+/-21). The number of eosinophils increased after inoculation of SP (p<0.05 vs. PBS, SM and EY). All treatments increased expression of interleukins (IL)-1beta and 6, tumor necrosis factor-alpha (TNF-alpha) and cyclooxgygenase-2 (COX-2) in the endometrium compared to pre-experiment values. Expression of COX-2 mRNA was significantly higher after infusion of SM than after PBS treatment (p<0.04). In conclusion, extender alone as well as seminal plasma and PBS causes an inflammatory endometrial response with the least pronounced response induced by EY-based semen extender.

Embryo transfer induces a subclinical endometritis in recipient mares which can be prevented by treatment with non-steroid anti-inflammatory drugs.

We tested the hypothesis that subclinical endometritis occurs after embryo transfer (ET) in the horse. Recipient mares were treated with meclofenamic acid (M) or flunixin meglumin (F) after ET or were left untreated (n=9 per group). Embryos were re-collected 4 days after transfer. Endometrial biopsies were taken for histology and analysis of cyclooxygenase-2 (COX-2) by immunohistochemistry and for PCR. Bacteriological swabs were collected from the uterus and lavage fluid of donor and recipient mares. Progesterone and prostaglandin F(2alpha) release was analysed in recipient mares after ET. Four days after ET, four embryos were recovered from group M and three from group F and untreated mares, each. The number of polymorph nuclear neutrophils was reduced in treated mares (p<0.05). Expression of mRNA for inflammatory cytokines did not differ between groups. In group M, expression of endometrial prostaglandin-E-synthase was higher than in group F (p<0.05). Three out of nine control mares underwent preterm luteolysis (p<0.05 vs. treatment groups), prostaglandin release (p<0.05) and the number of COX-2 positive cells (p<0.01) were significantly higher than in treated mares. Only few bacteriological swabs were positive. In conclusion, treatment of embryo recipient mares with non-steroid anti-inflammatory drugs inhibits the inflammatory response of the endometrium after ET. Meclofenamic acid may have advantages in comparison to flunixin meglumin due to a different influence on prostaglandin synthesis that may not result in inhibition of embryonic mobility.

M gene analysis of atypical strains of feline and canine coronavirus circulating in an Austrian animal shelter.

Coronavirus-positive samples of faeces collected in an Austrian animal shelter from 12 cats and 10 dogs were analysed by reverse transcriptase-pcr with primers amplifying a segment of the M protein gene, and by sequence analysis. In addition, the samples were subjected to S gene typing, using primers that differentiated between feline coronavirus (fcov) types I and II. A phylogenetic analysis of the M gene sequences revealed not only clearly segregating canine coronavirus (ccov) in the dogs, typical ccov sequences and the recently described fcov-like ccov, but also at least two genetic clusters of fcov in the cats, one species-specific, the other more closely related to fcov-like ccov. The M gene sequences of these new feline strains had at most 88 per cent identity with the fcov-like ccov strain 259/01 and only up to 85 per cent with any fcov sequence available in GenBank. In the phylogenetic tree they occupy an intermediate position between feline and canine coronaviruses.

CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates.

Extended-spectrum beta-lactamase (ESBL) production was demonstrated in five independent, multidrug-resistant isolates of enteroaggregative Escherichia coli (EAEC) from the United Arab Emirates, representing 11.3% of the EAEC isolates recovered during 1 year. All five isolates carried the bla(CTX-M-15) and the bla(TEM-1) genes, the former positioned 48 bp downstream of an ISecp1 element. In two isolates, the bla(CTX-M-15 )and bla(TEM-1) genes were located on a 95-kb plasmid. This is the first detailed description and characterisation of ESBL production in enteroaggregative E. coli and also the first report of CTX-M-producing organisms encountered on the Arabian Peninsula.

Pathology and viral distribution in fatal Usutu virus infections of birds from the 2001 and 2002 outbreaks in Austria.

In the summer of 2001, Usutu virus (USUV) was isolated for the first time in Europe, from an episode of mass mortality in Eurasian blackbirds (Turdus merula). In the present study, 40 of the birds (representing three species), confirmed as cases of USUV infection, were examined by four methods (histopathology, immunohistochemistry [IHC], in-situ hybridization [ISH] and reverse-transcriptase polymerase chain reaction [RT-PCR]). The major macroscopical finding was hepatosplenomegaly; histologically, neuronal necrosis, myocardial lesions, and coagulative necrosis of the liver and spleen were observed. IHC with cross-reactive polyclonal antibodies to West Nile virus detected viral antigen predominantly in brain neurons (40/40 birds; 100%), myocardial fibres (25/32; 78%), cells of the splenic capsule (29/33; 88%), renal glomeruli (22/35; 63%), tunica muscularis of intestines (17/22; 77%), proventricular glands (16/19; 84%), lungs (18/33; 55%) and hepatic Kupffer cells (7/38; 18%). ISH with an USUV-specific oligonucleotide probe demonstrated viral nucleic acid predominantly in brain neurons (40/40; 100%), myocardial fibres (24/33; 73%), splenic macrophages (12/34; 35%), renal tubular cells (19/36; 53%), tunica muscularis of intestines (13/32; 41%), proventricular glands (19/22; 86%), lungs (7/34; 21%) and hepatic Kupffer cells (12/38; 32%). All of 33 birds tested additionally by USUV-specific RT-PCR gave positive results.

Screening for West Nile virus infections of susceptible animal species in Austria.

Avian mortality and encephalomyelitis in equines are considered good indicators for West Nile virus (WNV) activity. We retrospectively tested 385 horse sera for WNV antibodies and looked for WNV nucleic acid and/or WNV antigen in paraffin embedded tissues from 12 horses with aetiologically unresolved encephalomyelitis and 102 free-living birds of different species which had been found dead. With the exception of four horses originating from eastern European countries investigated on the occasion of transit through Austria, all horse sera were negative. Nested RT-PCR of the horse tissues yielded no amplification of WNV-RNA. Also, all bird samples, examined by immunohistochemistry, in situ hybridization and nested RT-PCR were negative for WNV. These results indicate that currently WNV cannot be considered a significant pathogen in Austria.

Equine neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis (NCL) is an inherited, neurodegenerative disorder with fatal outcome in humans. It has also been described in some animal species; this is the first report of NCL in equines. Three horses showed developmental retardation, slow movements and loss of appetite at the age of six months. Neurological symptoms, as well as visual failure in one case, were noticed at the age of 1 year. Due to slowly progressing deterioration, euthanasia was indicated 1.5 years after onset of conspicuous behavior. At necropsy, slight flattening of the gyri and discoloring of the brain was noticed. Histopathology revealed eosinophilic, autofluorescent material in the perikarya of neurons throughout the brain and spinal cord. Identical material was found in neurons of retina, submucous and myenteric ganglia, as well as in glial cells. Immunohistochemistry, using antiserum against subunit c of mitochondrial ATP synthase, showed positive signals in neurons and glial cells. Electron microscopical studies revealed fingerprint profiles mixed with rectilinear structures in markedly enlarged lysosomes of neurons and renal tubules, and rectilinear structures mixed with curvilinear bodies in macrophages and lymphocytes of lymph nodes. Thus, our study presents the first occurrence of lysosomal storage disease in horses, further characterized by immunohistochemical and electron microscopical investigations as NCL.

Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR.

Sacbrood virus (SBV) infects larvae of the honeybee (Apis mellifera), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.

Isolation and characterization of a new subtype of Borna disease virus.

Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus Bornavirus of the family Bornaviridae. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.