First molecular detection and characterization of Akabane virus in small ruminants in Turkey.

Abortion outbreaks associated with congenital malformations in two distinct small-ruminant flocks were reported in Turkey in 2013-2014. This paper describes the first molecular characterization of Turkish Akabane virus strains in small-ruminant flocks using partial sequence analysis of the S segment and pathological findings.

West Nile Virus Infection in Horses: Detection by Immunohistochemistry, In Situ Hybridization, and ELISA.

This study describes the clinicopathologic findings in naturally occurring West Nile virus (WNV) infection in horses. WNV was diagnosed in a foal by immunohistochemical and in situ hybridization methods, and the presence of WNV antibodies was detected in 5 other horses with clinical signs suggestive of WNV infection. At necropsy of the foal, lymph nodes were edematous and enlarged, and the intestines showed diffuse congestion and focal hemorrhages. The most significant histologic lesions in this case were nonsuppurative meningoencephalomyelitis, particularly in the brainstem and spinal cord. Identification of viral RNA by in situ hybridization and viral antigen by immunohistochemistry was concentrated primarily in nerve fibers, glial cells, and their processes in brainstem and spinal cord and, to a lesser extent, within the cerebral hemispheres and cerebellum.

A Report on Bovine Ephemeral Fever Virus in Turkey: Antigenic Variations of Different Strains of EFV in the 1985 and 2012 Outbreaks Using Partial Glycoprotein Gene Sequences.

We described the aetiological agents of outbreaks of bovine ephemeral fever (BEF) that occurred in 1985 and 2012 in Turkey, and identify mutations in the viruses from both outbreaks. Outbreaks have emerged periodically every 4-5 years in the same regions in Turkey. Because these regions are located in a subtropical climatic zone, good conditions for vector populations exist. The results of this study show that the BEFVs from outbreaks in Turkey vary significantly. Effective prevention will require a vaccine that contains BEFVs from different genetic clusters.

Molecular characteristics of bovine virus diarrhoea virus 1 isolates from Turkey: approaches for an eradication programme.

Forty pestivirus isolates sampled from cattle in Turkey between 2002 and 2007 were characterized according to 5' untranslated region (5'UTR) sequences and autoprotease (N(pro) ) gene sequences. The sampling of Bovine virus diarrhoea viruses (BVDVs) from 15 farms in five different regions indicated that BVDV 1-l (18/40, 45%) was the predominant genotype in Turkey; the samples also contained the genotypes 1-f (10/40, 25%), 1-b (7/40, 17.5%), 1-d (3/40, 7.5%), and 1-a (2/40, 5%), respectively.

Dual infection of fetal and neonatal small ruminants with border disease virus and peste des petits ruminants virus (PPRV): neuronal tropism of PPRV as a novel finding.

Dual infection of 26 fetal and neonatal small ruminants with border disease virus (BDV) and peste des petits ruminants virus (PPRV) is reported. The animals included five aborted lamb fetuses, 19 neonatal lambs and two neonatal kids from flocks in regions of the Black Sea and the Aegean region. BDV and PPRV antigens were detected immunohistochemically in the brain, oral mucosa, intestine and lung of infected animals. Reverse transcriptase-polymerase chain reaction was used to demonstrate PPRV and BDV in samples of the spleen, lymph node, lung and brain from infected animals. On the basis of observations made, it is concluded that brain damage following intrauterine infection with BDV facilitates the passage of PPRV to the brain and results in infection of neuronal and glial cells by PPRV.

Neuropathologic study of border disease virus in naturally infected fetal and neonatal small ruminants and its association with apoptosis.

The present study describes the pathologic changes and cellular apoptosis in the central nervous system (CNS) of fetal and neonatal small ruminants infected with border disease virus (BDV), as demonstrated by immunohistochemistry and in situ hybridization. Abortions of ewes and goats were observed, as were births of lambs and kids with poor survival rates and nervous signs. Lesions included cerebellar hypoplasia, porencephaly, hydranencephaly, and nonsuppurative meningoencephalomyelitis with hypomyelinogenesis. Viral antigens and RNA were present in neuropil, glial, and neuronal cells, especially in periventricular areas, cerebellum, and brainstem. TUNEL positivity and labeling of anti-bax and anti-caspases 3, 8, and 9 were detected in BDV-infected CNSs, especially in glial and neuronal cells. The double immunostaining and TUNEL assay revealed that in BDV-infected animals, not only were BDV-infected glial and neuronal cells undergoing apoptosis, but so were uninfected cells in close vicinity of BDV-infected cells. The expression of activated caspases 3, 8, 9; bax; and TUNEL in glial and neuronal cells of the infected fetal and neonatal kids were significantly (P < .05) higher than those of the infected fetal and neonatal lambs. Yet, the expression of bcl-2 in the CNSs of the infected fetal and neonatal lambs was higher (P < .05) in neuronal and glial cells than in those of the infected fetal and neonatal kids. The results suggest that cell death in the BDV-infected CNS is induced by intrinsic and extrinsic cascades of apoptotic pathways.

The detection and genetic characterization based on the S1 gene region of BCoVs from respiratory and enteric infections in Turkey.

We investigated bovine coronavirus (BCoV) as an etiological agent in cattle with clinical respiratory and digestive signs using 147 feces and 199 nasal swab samples. A total of 18 test samples (16 feces and 2 nasal swap samples) were detected positive by ELISA and/or RT-PCR targeting the BCoV N gene. The partial S1 gene regions of BCoVs (An-4 and An-11) detected in feces samples from two herd-mate dairy calves were compared. Virological and serological results indicated that BCoVs are widespread in Turkey and are likely etiological agents in diarrhea cases in calves.

Detection and sequence analysis of equine gammaherpesviruses from horses with respiratory tract disease in Turkey.

The equid herpesvirus 2 (EHV-2) and 5 (EHV-5), identified agents of respiratory infections and keratoconjunctivitis cases in some equids, comprise a high degree of antigenic heterogeneity. Prevalence and genetic characterization of EHV-2 and EHV-5 strains from Turkey were investigated in this study. A total of 73 nasal swabs and 54 blood specimens were sampled from horses with respiratory tract diseases characterized by mucopurulent nasal discharge and occasional coughing. Overall, EHV-2- and EHV-5-specific DNA amplicons were obtained from 19.2% (14/73) and 21.9% (16/73) of horses tested by multiplex nested PCR. Sequences of EHV-2 and EHV-5 glycoprotein B (gB) gene were used in a phylogenetic analysis that included six EHV-2 and three EHV-5 isolates, which showed that the Turkish EHV-2 and EHV-5 strains have marked sequence divergence from European strains and from each other. Turkish EHV-2 isolates were divided into two distinct subdivisions, and a few isolates were located on a separate branch. This study provides the first epidemiological and phylogenetical report about EHV-2 and EHV-5 infections in Turkey.

Border disease virus (BDV) infections of small ruminants in Turkey: a new BDV subgroup?

Blood samples from sheep and/or goats from eight small ruminant flocks in the Turkish provinces of Aydin and Burdur were tested for the presence of Pestiviruses using an antigen-capture ELISA. From clinically affected animals, pathological and immunohistochemical findings were recorded. Post mortem examination of a virus-positive lamb showing abnormal fleece and paralysis of the hind legs revealed nonsuppurative meningoencephalomyelitis with hypomyelinogenesis. By immunohistochemistry Pestivirus antigen was detected in all parts of the brain including cerebellum, cerebral hemispheres and midbrain. Two Pestivirus isolates from a sheep and a goat kid, respectively, were isolated from samples that were positive in the antigen-capture ELISA. Genetic typing using the 5'-NTR (288bp) and N(pro) (738bp) showed that both were Border disease virus (BDV) isolates. By phylogenetic analysis, they formed a cluster clearly separated from the known clusters BDV-1 to BDV-6 and might therefore represent a new subgroup (BDV-7?). This is the first report confirming the occurrence and partial characterisation of BDV infection in small ruminants in Turkey.

Visceral leishmaniosis and parapoxvirus infection in a Mediterranean monk seal (Monachus monachus).

A Mediterranean monk seal was shown by immunohistochemical and polymerase chain reaction techniques to be dually infected with a Leishmania sp. and parapoxvirus. The pathological findings included a deep ulcer on the side of the head, ulcers on the gingival and inner aspect of the lower lip, enlarged lymph nodes and tonsils, and respiratory lesions (pulmonary consolidation, oedema, haemorrhages and emphysema; tracheal and bronchial congestion, exudates and haemorrhage). Amastigotes were demonstrated in macrophages in the lymph nodes and spleen, and intracytoplasmic inclusion bodies were observed in the tracheal and oral mucosa.

A sheeppox virus outbreak in Central Turkey in 2003: isolation and identification of capripoxvirus ovis.

Poxvirus epidemics occur almost every year and cause significant economic losses for small-scale animal producers in Turkey. In this study, the causative agent of the most recent epidemic in Central Anatolia was detected in clinical samples using electron microscopy (EM) and amplified using an in house polymerase chain reaction procedure for the first time. Additionally, the aetiological agent was isolated from a sheep and identified using EM and PCR.

[Kinetics and persistence of neutralizing antibodies against bovine viral diarrhea virus-1 and -2 and border disease virus after two step vaccination of cattle]. / Kinetik und Persistenz neutralisierender Antikörper gegen das Bovine Virusdiarrhoe Virus-1 und 2 und das Border Disease Virus nach zweistufiger Impfung von Rindern.

The aim of the study was the assessment of rise and persistence of neutralizing antibodies (nAb) to bovine viral diarrhea virus (BVDV) and border disease virus (BDV) after a two step vaccination using an inactivated BVDV/BDV (Mucobovin) and a modified live BVDV vaccine (Vacoviron). In a first experiment eight heifers were kept in isolation and were serologically surveyed regularly over a three year period after vaccination. The same experiment was done with 80 vaccinated cattle kept under field conditions. Neutralizing antibody titres were monitored using homologous as well as heterologous BVDV and one BDV strain, respectively. Maximum titres were obtained two to three months after vaccination. During the three years of monitoring the antibody titres decreased but never fell below the detection limit. This slow antibody regression demonstrates that a single two step vaccination elicited high nAb titres which persist over at least three years. These results might serve as a decision tool when considering the necessity and time of revaccination of cattle, which have been vaccinated using the two step method.

Foetal protection against bovine virus diarrhoea virus after two-step vaccination.

In order to assess the efficacy of a two-step vaccination protocol with respect to foetal protection against transplacental infections with bovine virus diarrhoea virus (BVDV) with special attention to BVDV-2 seronegative heifers were vaccinated with an inactivated BVDV-1 vaccine and boostered with a modified live BVDV-1 vaccine after 4 weeks. A second group was left unvaccinated as control. Between days 30 and 120 of pregnancy the heifers of both groups were intranasally challenged with a mixture of BVDV-1 and -2. All heifers of the vaccinated group gave birth to nine clinically healthy, seronegative (precolostral) and BVDV-free calves. In contrast in the control group four BVDV viraemic underdeveloped calves were born. Additionally, one calf was stillborn and another viraemic calf was not viable and died 2 days after birth. All six calves of the control group were viraemic with BVDV-2. This study demonstrated for the first time that two-step vaccination of breeding cattle with a modified live BVDV vaccine 4 weeks after application of an inactivated BVDV vaccine was capable of providing a foetal protection against transplacental infection with BVDV-2.

[Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm]. / Zur Differentialdiagnostik Klassische Schweinepest/Border Disease: Seroepidemiologische Untersuchung einer Pestivirusinfektion auf einem schaf- und schweinehaltenden Betrieb.

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.

Virological and serological studies on the role of PI-3 virus, BRSV, BVDV and BHV-1 on respiratory infections of cattle. I. The detection of etiological agents by direct immunofluorescence technique.

Nasal cells extracted from nasal swabs obtained from 95 cattle with signs of respiratory disease, out of eleven different herds, were tested for BHV-1, PI-3 virus, BRSV and BVDV using direct immunofluorescence technique. Viral antigen positive samples were detected in seven out of eleven herds examined. Of the 95 individual diseased cattle, 19 were found positive for at least one viral antigen. It was found that especially BHV-1 and PI-3 virus are important causative agents in cattle respiratory disease, both or in combination with other pathogenic agents. Multiple infection in virologically positive herds were observed in six (9.8%) of 61 animals tested. The findings reveal that single or multiple infections of selected viruses may be present in an important range in cattle and that direct immunofluorescence technique as a rapid method, based on the detection of viral antigen in nasal swab samples, is useful to establish the viral aetiology of acute bovine respiratory disease caused by these viruses, particularly in the diagnosis of mixed viral infections.