Slime-producing staphylococci as causal agents of subclinical mastitis in sheep.

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection.

Rabies outbreak in Greece during 2012-2014: use of Geographical Information System for analysis, risk assessment and control.

The objectives of this work were (i) geographical analysis of the 2012-2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (<343·5 m), within a distance of 1 km from human settlements. Median distance from livestock farms was 201·25 m. Most people potentially exposed to rabies (889/1060) presented with dog bite injuries. Maximum entropy analysis revealed that distance from farms contributed the highest percentage in defining environmental niche profiles for rabid foxes. Oral vaccination programmes were implemented in 24 administrative units of the country during 2013 and 2014, covering a total surface area of ~60 000 km2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity.

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine.

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian-like H1N1 and two reassortant H1N2 and H3N2 viruses with human-origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross-reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.

First evidence of Leishmania infection in European brown hare (Lepus europaeus) in Greece: GIS analysis and phylogenetic position within the Leishmania spp.

Although the existence of a sylvatic transmission cycle of Leishmania spp., independent from the domestic cycle, has been proposed, data are scarce on Leishmania infection in wild mammals in Greece. In this study, we aimed to investigate the presence of Leishmania infection in the European brown hare in Greece, to infer the phylogenetic position of the Leishmania parasites detected in hares in Greece, and to identify any possible correlation between Leishmania infection in hares with environmental parameters, using the geographical information system (GIS). Spleen samples from 166 hares were tested by internal transcribed spacer-1 (ITS-1)-nested PCR for the detection of Leishmania DNA. Phylogenetic analysis was performed on Leishmania sequences from hares in Greece in conjunction with Leishmania sequences from dogs in Greece and 46 Leishmania sequences retrieved from GenBank. The Leishmania DNA prevalence in hares was found to be 23.49 % (95 % confidence interval (CI) 17.27-30.69). The phylogenetic analysis confirmed that the Leishmania sequences from hares in Greece belong in the Leishmania donovani complex. The widespread Leishmania infection in hares should be taken into consideration because under specific circumstances, this species can act as a reservoir host. This study suggests that the role of wild animals, including hares, in the epidemiology of Leishmania spp. in Greece deserves further elucidation.

Dissemination of intestinal pathogens between lambs and puppies in sheep farms.

Objectives of the present work were (i) to confirm pathogens implicated in cases of diarrhoea in newborn and young lambs in sheep farms in Greece and (ii) to investigate a possible relation in dissemination of pathogens between lambs and dogs present in the farm. Work was carried out in 22 sheep farms, with (i) flock size over 150 animals, (ii) presence of clinical signs of diarrhoea in lambs in the flock and (iii) close and continuous contact and movement of shepherd dogs within the animal shed of each farm. Faecal sample collection from lambs was performed within 48 h of onset of clinical signs and prior to administration of any antimicrobial or antiparasitic medication to lambs. Faecal samples were also collected from puppies in the farm. In total, samples were collected from 126 lambs and 58 puppies. Samples were processed by using established techniques for isolation of bacteria, detection of viruses and observation of protozoan oocycts. Escherichia coli isolates obtained during the study, were tested for antimicrobial resistance against a variety of antimicrobial agents. In total, 236 bacterial isolates were recovered from faecal samples of lambs and 165 isolates from faecal samples of puppies. E. coli was the most frequently isolated microorganism: 104 isolates from lambs and 109 isolates from puppies were recovered. Other bacteria isolated were Enterobacter spp., Proteus spp., Klebsiella spp., (lambs and puppies), Clostridium perfringens, Citrobacter freundi, Salmonella enterica subsp. diarizonae (only lambs) and Streptococcus spp. (only puppies). Group A Rotavirus was detected in samples from lambs (2.5%) and Parvovirus in samples from puppies (5%). Cryptosporidium spp. oocysts were observed in samples from lambs and puppies. This is the first report of isolation of S. enterica subsp. diarizonae and of detection of Rotavirus from lambs in Greece. Rates of E. coli isolates from puppies resistant to antimicrobial agents were, in general, smaller than respective rates in isolates from lambs. Two pairs of isolates from the same farm (one from a lamb and one from a puppy) with identical patterns of resistance to antimicrobial agents were detected, which provides some evidence in support of a hypothesis that members of each pair might possibly have been spread from one animal species to the other.

Experiences from the 2014 outbreak of bluetongue in Greece.

Objective of this paper was to review relevant work and to present a general account of the bluetongue outbreak, which occurred in Greece in 2014. In total, 2895 outbreaks of the disease have been reported by the veterinary authorities of Greece; sheep, goats and cattle were affected with officially reported morbidity rates of 11.0%, 2.0% and 3.5%, respectively. No vaccinations were allowed and conservative measures were implemented to attempt to limit the disease, which at the end had expanded throughout the country. In field investigations, a significantly higher bluetongue morbidity rate (27.5%) in sheep has been reported. During that work, clinical anaemia was encountered, which was characterised as macrocytic, hypochromic, regenerative and non-haemolytic. Other investigations, which are reviewed in this paper, have described an outbreak of Citrobacter freundii-associated enteritis in newborn kids, offspring of goats subclinically infected with Bluetongue virus, increased rate of early embryonic deaths, reduced conception rates, increased incidence risk of mastitis and reduced milk yield in herds of subclinically-infected cattle and detection of the virus from hunter-harvested tissue samples of roe-deer. In 2015, vaccines against the disease have been licenced; vaccinations started in May 2015. Then, in 2015, only one outbreak of the disease was confirmed, which could have been the result of a combination of reasons acting concurrently to prevent further cases.

A serosurvey for selected pathogens in Greek European wild boar.

OBJECTIVES: Serum samples, collected from 94 European wild boar (Sus scrofa) during the hunting seasons 2006 -2010 from different regions of Greece, were examined in order to estimate the role of these wildlife species as reservoir of pathogens important for livestock and/or public health. MATERIALS AND METHODS: The assays used for this purpose were commercial indirect ELISA for the detection of antibodies against porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome (virus) (PRRSV), Aujeszky's disease virus (ADV), influenza A (IA) virus, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Salmonella species, Trichinella species and indirect immunofluorescence antibody test for the detection of antibodies against Toxoplasma gondii and Neospora caninum. RESULTS: Antibodies against PCV-2, PRRSV, ADV, IA virus,A. pleuropneumoniae, M. hyopneumoniae, Salmonella species, Trichinella species, T. gondii and N. caninum were detected in 19.1 per cent, 12.8 per cent, 35.1 per cent, 1.1 per cent, 57.4 per cent, 0 per cent, 4.3 per cent, 6.4 per cent, 5.2 per cent and 1.1 per cent of the samples, respectively. Cluster analysis revealed a hot spot of seropositivity near Bulgarian border; seropositivity to ADV was more common among female animals. CONCLUSIONS: These results indicate exposure of wild boar to most of the above-mentioned pathogens, raising concern about the possibility that these species may pose a significant health risk for livestock and/or humans.

Orf virus infection in sheep or goats.

Orf virus, a member of the genus Parapoxvirus, is the causative agent of contagious ecthyma ('Orf'). It is a pathogen with worldwide distribution, causing significant financial losses in livestock production. The disease mainly affects sheep and goats, but various other ruminants and mammals have been reported to be infected as well. It is also a zoonotic disease, affecting mainly people who come in direct or indirect contact with infected animals (e.g. farmers, veterinarians). The disease is usually benign and self-limiting, although in many cases, especially in young animals, it can be persistent and even fatal. Production losses caused by Orf virus are believed to be underestimated, as it is not a notifiable disease. This review of literature presents all latest information regarding the virus; considerations regarding treatment and prevention will be also discussed.

Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010.

A West Nile virus (WNV) lineage 2 strain was molecularly identified and characterised in a Eurasian magpie hunted in Greece in 2010, during a WNV outbreak in humans. Phylogenetic analysis revealed the highest sequence similarity (>99%) with other WNV lineage 2 strains derived from birds of prey in Austria and Hungary (2004­2009). This first molecular detection of WNV in sedentary wild birds in Greece, which are possible reservoirs of the virus, is a public health concern.

Major histocompatibility complex variation at class II DQA locus in the brown hare (Lepus europaeus).

The major histocompatability complex (MHC) is a multigene family of receptors that bind and present antigenic peptides to T-cells. Genes of the MHC are characterized by an outstanding genetic polymorphism, which is considered to be maintained by positive selection. Sites involved in peptide binding form binding pockets (P) that are collectively termed the peptide-binding region (PBR). In this study, we examined the level of MHC genetic diversity within and among natural populations of brown hare (Lepus europaeus) from Europe and Anatolia choosing for analysis of the second exon of the DQA locus, one of the most polymorphic class II loci. We aimed at an integrated population genetic analysis of L. europeaus by (i) correlating MHC polymorphism to genetic variability and phylogenetic status estimated previously from maternally (mtDNA) and biparentally (allozymes, microsatellites) inherited loci; and (ii) comparing full-length exon amino acid polymorphism with functional polymorphism in the PBR and the binding pockets P1, P6 and P9. A substantial level of DQA exon 2 polymorphism was detected with two completely different set of alleles between the Anatolian and European populations. However, the phylogeny of full-length exon 2 Leeu-DQA alleles did not show a strong phylogeographic signal. The presence of balancing selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous in the PBR and a trans-species pattern of evolution detected after phylogenetic reconstruction. The differentiating patterns detected between genetic and functional polymorphism, i.e. the number and the distribution of pocket variants within and among populations, indicated a hierarchical action of selection pressures.

Atypical PrPsc distribution in goats naturally affected with scrapie.

The brain and spinal cord of 48 goats from two Greek herds in which scrapie had been reported were examined. All animals were symptomless at the time of euthanasia. Notably, no lesions were observed either at the level of the obex or at other regions of the brain and spinal cord. Immunohistochemical examination revealed PrPsc labelling of the linear and fine punctuate types, mainly in the cerebral cortices, of 36 goats. Twenty-seven of them were negative by ELISA (designed to detect proteinase-resistant PrP) at the level of the obex but positive in a pooled brain sample, and the majority carried PrP genotypes associated with scrapie susceptibility. Surprisingly, in 16 of the 27 animals, PrPsc deposits were detected only in the rostral parts of the brain. In addition, nine animals which were ELISA-positive at the level of the obex exhibited positive immunoreactivity, but not in the dorsal vagal nucleus. The findings indicate that this unusual scrapie type may have been underdiagnosed previously and may be of importance in scrapie surveillance programmes.

Histopathological and immunohistochemical features of natural goat scrapie.

Histopathological and immunohistochemical examinations were performed on the brain and spinal cord of 37 goats from two Greek herds in which scrapie had been reported. Of the 37 animals, 18 were from a herd consisting only of goats and 19 were from a herd of goats mixed with sheep. The goats studied were grouped on the basis of the presence or absence of clinical signs. Distinctive lesions and PrP(sc) (PrP, prion protein) deposition were found in the central nervous system (CNS) of eight clinically affected animals and six symptomless animals. The lesion profile and PrP(sc) distribution varied both between and within groups, variation being particularly pronounced in the symptomless goats. The results concerning the latter group suggested a poor correlation between the intensity of lesions, the amount of PrP(sc) in the CNS, and the manifestation of clinical signs. Immunohistochemical examination revealed 10 different PrP(sc) types, four of which are reported for the first time in goats. All scrapie-affected animals carried the VV(21)II(142)HH(143)RR(154) genotype, with the exception of two goats that carried the HR(143) dimorphism and had detectable PrP(sc) deposits. The results suggest that the histopathological and immunohistochemical profile of the natural disease in goats is influenced by the PrP genotype and age of the animals but may not be directly associated with the presence or otherwise of clinical signs.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in mice.

Mice (n=20) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 18-59 days post-infection (dpi), except for two mice that died at 6 and 32 dpi, and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical examination. Apart from the two deaths, the experimental infection was inapparent, but virus was invariably recovered from faeces and several organs. The main histopathological lesions were focal interstitial pancreatitis, depletion of thymus and Peyer's patches, and interstitial pneumonia. Additionally, in the two mice that died, multifocal interstitial myocarditis was observed. EMCV antigen was detected in the cytoplasm of pancreatic acinar cells and in macrophages of the lung and the thymus. Antigen was also detected in the cytoplasm of cardiac muscle cells from three animals, including the two that died. The results support the role of mice, in addition to rats, as reservoir hosts in the epidemiology of EMCV infections on pig farms.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats.

Rats (n=40) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 11-62 days post-infection (dpi) and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical evaluation. This experimental infection was inapparent, but virus was isolated from faeces and several organs of all animals. The main histopathological changes were focal interstitial pancreatitis, degeneration and necrosis of pancreatic acinar cells, depletion of thymus and Peyer's patches, and interstitial pneumonia. EMCV antigen was detected in the cytoplasm of cardiac muscle cells, pancreatic acinar cells and hepatic epithelial cells, and in macrophages of the spleen, lung and thymus. In the heart (the target organ of EMCV in pigs), the presence of EMCV in cardiac muscle cells without lesions lends support to the hypothesis that the rat is a natural reservoir host species of EMCV. The persistence of virus in the macrophages of the thymus may represent a mechanism of perpetuation and reactivation, under immunosuppressive conditions, of the infection.

Prevalence of BVDV infection in Greek dairy herds.

Thirty-nine Greek dairy herds, totalling 6333 cattle, enrolled in a voluntary bovine viral diarrhoea virus (BVDV) eradication programme based on the identification and removal of persistently infected (PI) animals. The aim of this study was to estimate the prevalences of BVD antigen-positive and PI animals, and investigate the significance of the associations between the prevalence estimates and herd size. Initially, all animals were bled and examined for BVDV, using an antigen ELISA. A second sample was collected from the positive animals, after a period of at least three weeks. Animals retested positive were classified as PI. Antigen positive and PI animals were detected in all herds. The respective mean prevalences, adjusted for the test's accuracy and the herd-clustering effect, were 14% (95%CI: 11-18%) and 1.3% (0.8-1.8%), respectively. Herd size was not associated with the prevalence of antigen-positive or PI animals.

Effect of challenge dose and age in experimental infection of pigs with encephalomyocarditis virus.

Two experiments were performed to compare the severity of encephalomyocarditis virus (EMCV) infection in pigs. The pigs were challenged with the Greek myocardial strain, at different ages and with different doses. In the first experiment, nineteen susceptible pigs, 40 days old, were divided into three groups and were experimentally infected with 10(6) TCID(50), 10(4) TCID(50) or 10(2) TCID(50) of the Greek EMCV strain. In the second experiment, 10 susceptible pigs, of either 20 or 105 days, were divided into two groups according to age and were experimentally infected with 10(6) TCID(50) of the Greek EMCV strain. In addition, five piglets, each one the same age as its experimental group, were used as uninfected controls. No clinical signs were observed after infection, except a transient temperature rise in some pigs. Another important observation was the difference in mortality between groups. The survival rate of the 40-day-old pigs was inversely related to the viral dose. In these pigs, a positive association between the viral dose and the severity of macroscopical and histopathological lesions of the heart was also evident. Viral isolations from various organs of the challenged 40-day-old pigs increased with the increasing dose level. When challenged with 10(6) TCID(50) of EMCV, there was no difference in the fatality rate of the 20- and 40-day-old pigs, but none of the 105-day-old pigs died. The severity of the macroscopical and the histopathological heart lesions was inversely related to the age of the pigs. Furthermore, viral isolations from the various organs were higher in 20- and 40-day-old pigs than in the older ones. In 40-day-old pigs, neutralizing antibodies linearly increased as the dose increased. These antibodies were consistently lower in 20-day-old pigs. Viraemia, and nasal and faecal excretions were detected in all groups and lasted 1-3 days, except for the 105-day-old pigs whose symptoms lasted for an additional day.

Equine infectious anemia in mules: virus isolation and pathogenicity studies.

There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A and developed signs of the disease; they were euthanatized or died at day 22 and 25 post-inoculation, respectively. Mule E served as a negative control. The virus was isolated from the plasma samples of mules with clinical signs of the disease (A, C and D), but not from the asymptomatic mule B. Both proviral DNA and viral RNA were amplified from blood and tissues of the infected animals by nested polymerase chain reaction (nPCR). Antibodies were not detected in the two experimentally infected mules until their natural death or euthanasia. Clinicopathological and laboratory findings showed that, in mules, EIAV produced clinical signs similar to those observed in horses and ponies. Nested PCR proved to be a rapid, sensitive and specific diagnostic method for the detection of EIAV, regardless of the disease stage.

Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods.

A duplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bluetongue virus (BTV) in clinical samples was developed. This assay, which detects the highly conserved S10 region of BTV, was assessed for sensitivity and application as a rapid and dependable diagnostic tool by comparison with standard assays of virus detection, such as virus isolation in embryonated chicken eggs and cell culture. Simultaneous detection of BTV and host beta-actin RNAs minimizes the possibility of false negative results. The sensitivity of the assay was found to be equal to five cell culture infectious dose (CCID(50)) units and its specificity was confirmed as no RT-PCR product was detected with RNAs from two closely related orbiviruses, i.e. epizootic haemorrhagic disease virus (serotypes 1, 2 and 318) and African horse sickness virus, serotype 9, or RNAs from uninfected BHK-21 cells and blood samples from uninfected sheep or goats. In this study, 36 blood samples from naturally infected mixed flocks of sheep and goats were examined. Seventeen animals were identified as BTV-positive by RT-PCR, whereas only 13 were found positive by virus isolation in embryonated chicken eggs and nine by cell culture assays. These results indicate that the duplex RT-PCR could be a useful technique for monitoring BTV infection in the field.