Prevalence of equine gammaherpesviruses on breeding farms in Turkey and development of a TaqMan MGB real-time PCR to detect equine herpesvirus 5 (EHV-5).

Equine herpesvirus type 2 (EHV-2) and EHV-5 are members of the subfamily Gammaherpesvirinae. The viruses are detected in horses with upper respiratory tract disease and are associated with low performance in racehorses. The aim of the current study was to use nested PCR to investigate the epidemiology of EHV-2 and EHV-5 in Arabian horse populations from breeding farms located in three different cities (Eskisehir, Malatya, and Bursa) in Turkey, using a real-time quantitative PCR (qPCR) with a TaqMan® minor-groove-binder (MGB) probe to detect EHV-5. Screening of blood and ocular and nasal swab samples by nested PCR showed the prevalence of EHV-2 and EHV-5 to be 59 % and 62 %, respectively, with a coinfection rate of 45 %. Thirty-seven isolates from blood samples were identified as EHV-2 using nested PCR. To develop the EHV-5 qPCR, a pair of primers and an MGB probe were designed based on a highly conserved genomic region encoding glycoprotein B (gB). The detection limit of the qPCR was 10 molecules per reaction, and it specifically detected EHV-5 and no other herpesviruses infecting horses (EHV-1, EHV-2, or EHV-4). When applied to field samples, the assay proved to be more sensitive than a well-established nested PCR. Therefore, the qPCR developed in this study provides a rapid, reliable, and sensitive diagnostic assay for the detection of EHV-5, and it complements other diagnostic procedures for equine respiratory disease.

[Infection control and hygiene management in equine hospitals]. / Infektionsprävention und Hygiene-management in Pferdekliniken.

With the rising importance of nosocomial infections in equine hospitals, increased efforts with regard to biosecurity and infection control are necessary. This even more since nosocomial infections are often associated with multi-drug resistant pathogens. Consequently, the implementation of targeted prevention programs is essential. Since nosocomial infections are usually multifactorial events, realization of only a single measure is rarely effective to overcome nosocomial spread in clinical practice. Equine patients may be colonized at admission with multi-drug resistant pathogens such as methicillin resistant Staphylococcus aureus (MRSA) and/or extended spectrum beta lactamase-producing (ESBL-) Enterobacteriaceae. Regardless of their individual resistance properties, these bacteria are common and usually unnoticed colonizers of either the nasopharynx or the intestinal tract. Also viral diseases caused by equine herpesvirus 1 (EHV-1) and EHV-4 may reach a clinic by patients which are latently infected or in the incubation period. To prevent nosocomal outbreaks, achieve an interruption in the infection chain and to eradicate infectious agents from the hospital environment, a professional hospital management is necessary. This should be adapted to both the wide range of pathogens causing nosocomial infections and the individual needs of equine patients. Amongst others, this approach includes a risk classification of equine patients at admission and information/enlightenment of the animal owners at discharge. An efficient management of inpatients, a targeted hygiene management and clear responsibilities with respect to biosecurity together with a surveillance of nosocomial infections form the cornerstone of infection control in equine hospitals.

Long-lasting airway inflammation associated with equid herpesvirus-2 in experimentally challenged horses.

The aim of this trial was to investigate the putative involvement of equid herpesvirus 2 (EHV-2) in airway inflammation of adult horses. Six horses received corticosteroid treatment, before either mock infection (n=2) or EHV-2 strain LK4 inoculation (n=4). These four horses were also submitted to immunosuppression 84 days post inoculation. EHV-2 was detected by quantitative PCR in respiratory samples up to respectively 21 days and 14 days. Nested PCR, cloning and sequencing allowed the detection of five different 'field' strains throughout the trial. Neutrophils proportions were transiently increased in respiratory fluids; neutrophilia being significantly associated with concomitant EHV-2 detection. The laboratory findings reproduced in this trial were compatible with sub-clinical lower airway inflammation and suggest that EHV-2 infection should be suspected when investigating poorly-performing horses.

Isolation and characterisation of equine influenza viruses (H3N8) from Europe and North America from 2008 to 2009.

Like other influenza A viruses, equine influenza virus undergoes antigenic drift. It is therefore essential that surveillance is carried out to ensure that recommended strains for inclusion in vaccines are kept up to date. Here we report antigenic and genetic characterisation carried out on equine influenza virus strains isolated in North America and Europe over a 2-year period from 2008 to 2009. Nasopharyngeal swabs were taken from equines showing acute clinical signs and submitted to diagnostic laboratories for testing and virus isolation in eggs. The sequence of the HA1 portion of the viral haemagglutinin was determined for each strain. Where possible, sequence was determined directly from swab material as well as from virus isolated in eggs. In Europe, 20 viruses were isolated from 15 sporadic outbreaks and 5 viruses were isolated from North America. All of the European and North American viruses were characterised as members of the Florida sublineage, with similarity to A/eq/Lincolnshire/1/07 (clade 1) or A/eq/Richmond/1/07 (clade 2). Antigenic characterisation by haemagglutination inhibition assay indicated that the two clades could be readily distinguished and there were also at least seven amino acid differences between them. The selection of vaccine strains for 2010 by the expert surveillance panel have taken these differences into account and it is now recommended that representatives of both Florida clade 1 and clade 2 are included in vaccines.

Pathogenesis of equine herpesvirus-1 infection in the mouse model.

Equine herpesvirus-1 (EHV-1) is a major equine pathogen causing respiratory diseases, abortions and severe neurological disorders. The basis of neurological disturbances is, as in other organs, infection of endothelial cells, followed by vasculitis, thrombosis and ischaemic damage of the parenchyma. Here, a murine model was used to explore the mechanism of entry to, and spread within the brain, the cell affinity of the agent and the modulating role of the immune defence, which are all factors governing the pathogenesis of the neurological disease. Because controversial views exist about these mechanisms, we undertook a neuropathological study with intranasally infected adult mice. EHV-1 entered the brain through the olfactory neuroepithelium and along the olfactory nerves, and spread transsynaptically in rostro-caudal direction, using olfactory and limbic neuronal networks. Exclusively neurons were infected. The cellular immune reaction exerted a restraining effect on virus dissemination. Following nasal infection, the olfactory route was the major pathway for virus entry and dissemination, involvement of the trigeminal nerve in virus spread seems much less probable. In the adult mouse brain EHV-1 behaves as a typical neurotropic agent, using, similarly to other herpesviruses, the neuronal networks for dissemination. Vasculitis, the predominant type of lesion in natural infection, and endothelial cell positivity for EHV-1 were detectable only in the lung. Thus, this agent exhibits in the mouse a dual affinity: it is neurotropic in the brain, and endotheliotropic in visceral organs. Consideration of pathogenetic aspects of equine and experimental murine EHV-1 infections also helps a better understanding of human herpetic brain disease.

Detection of Equid herpesvirus 9 DNA in the trigeminal ganglia of a Burchell's zebra from the Serengeti ecosystem.

Equid herpesvirus 9 (EHV-9) was isolated from a herd of Thomson's gazelles affected by encephalitis. The natural host of EHV-9 is unknown, but zebras are suspected to be the source of infection in gazelles. To prove this hypothesis, we analyzed 43 sera from Burchell's zebras (Equus burchelli) and 21 Thomson's gazelles (Gazella thomsoni) from the Serengeti ecosystem for neutralizing antibodies. Seven zebra sera were positive for EHV-1, EHV-9 and EHV-1 from Grevy's zebra strains T965 and T616. The trigeminal ganglia of 17 other Burchell's zebras and one Thomson's gazelle were tested by EHV-9 gB and EHV-1 ICP0-specific nested polymerase chain reaction (PCR). PCR sequencing confirmed that one zebra ganglion was positive for EHV-9. These results suggest that the Burchell's zebras were exposed to EHV-9 and latently infected.

Sequence analysis of the equid herpesvirus 2 chemokine receptor homologues E1, ORF74 and E6 demonstrates high sequence divergence between field isolates.

Equid herpesvirus 2 (EHV-2), in common with other members of the subfamily Gammaherpesvirinae, encodes homologues of cellular seven-transmembrane receptors (7TMR), namely open reading frames (ORFs) E1, 74 and E6, which each show some similarity to cellular chemokine receptors. Whereas ORF74 and E6 are members of gammaherpesvirus-conserved 7TMR gene families, E1 is currently unique to EHV-2. To investigate their genetic variability, EHV-2 7TMRs from a panel of equine gammaherpesvirus isolates were sequenced. A region of gB was sequenced to provide comparative sequence data. Phylogenetic analysis revealed six 'genogroups' for E1 and four for ORF74, which exhibited approximately 10-38 and 11-27 % amino acid difference between groups, respectively. In contrast, E6 was highly conserved, with two genogroups identified. The greatest variation was observed within the N-terminal domains and other extracellular regions. Nevertheless, analysis of the number of non-synonymous (d(N)) and synonymous (d(S)) substitutions per site generally supported the hypothesis that the 7TMRs are under negative selective pressure to retain functionally important residues, although some site-specific positive selection (d(N)>d(S)) was also observed. Collectively, these data are consistent with transmembrane and cytoplasmic domains being less tolerant of mutations with adverse effects upon function. Finally, there was no evidence for genetic linkage between the different gB, E1, ORF74 and E6 genotypes, suggesting frequent intergenic recombination between different EHV-2 strains.

Characterization of equid herpesvirus 1 (EHV-1) related viruses from captive Grevy's zebra and blackbuck.

Equid herpes virus 1 (EHV-1) related isolates from a captive blackbuck (strain Ro-1) and Grevy's zebra (strain T965) behaved similarly to EHV-1 and EHV-9 in respect to their host cell range. Restriction enzyme analysis and a phylogenetic tree confirmed that Ro-1 and T965 were identical and more closely related to EHV-1 than to EHV-9. Differences from EHV-1 became obvious firstly, by amino acid alignments revealing two unique substitutions in the gB protein of Ro-1 and T965. Secondly, an EHV-1 type-specific monoclonal antibody did not detect its antigen on Ro-1, T965 or EHV-9 infected cells by immunohistochemistry. The results support the view that Ro-1 and T965 isolates represent a distinct, previously unrecognized species of equid herpesviruses.

Antibodies against equine herpesviruses and equine arteritis virus in Burchell's zebras (Equus burchelli ) from the Serengeti ecosystem.

A total of 51 sera from a migratory population of Burchell's zebras (Equus burchelli) were collected in the Serengeti National Park (Tanzania) between 1999 and 2001 to assess levels of exposure to equine herpesvirus types 1, 2, 4, 9 (EHV-1, -2, -4, -9), EHV-1 zebra isolate T965, and equine arteritis virus (EAV). Using virus-specific neutralizing antibody tests, seroprevalence was high for EHV-9 (60% of 45), moderate for EAV (24% of 51), and lower for the EHV-1-related zebra isolate (17% of 41), EHV-1 (14% of 49), and EHV-4 (2% of 50). No evidence for exposure to EHV-2 was found (0% of 51). The high level of exposure to EHV-9 is interesting because evidence of infection with this virus has not been previously described in any wild equine population. Although the epidemiology of EHV-9 in Burchell's zebras is presently unknown, our results suggest that in East Africa, this species may be a natural host of EHV-9, a neuropathogenic virus that was only recently isolated from captive Thomson's gazelles (Gazella thomsoni) in Japan. There is currently no evidence that EHV-9 induced mortality in Burchell's zebras in the Serengeti, but because of the reported virulence of this virus for more susceptible species such as Thomson's gazelles, viral transmission from infected zebras to ungulates may result in mortality.

Characterisation of three equine influenza A H3N8 viruses from Germany (2000 and 2002): evidence for frozen evolution.

Reported here are the results of antigenic and genetic characterisation of equine influenza strains causing local outbreaks reported to the Equine Diagnostic Centre in Berlin, Germany. In 2000, equine influenza virus was detected in a nasal swab from a non-vaccinated horse using a rapid diagnostic kit, but was not successfully isolated. Partial direct sequencing of the haemagglutinin (HA1) gene, indicated that the virus was a European lineage H3N8 subtype strain representative of strains isolated in several European countries during 2000. In 2002, two equine influenza viruses were isolated from nasal swabs both taken from unvaccinated horses with acute respiratory symptoms housed at the same stables. Antigenic characterisation using a panel of ferret antisera suggested that these isolates also belonged to the European lineage of H3N8 viruses. Analysis of deduced HA1 amino acid sequences confirmed that the HA1 of both isolates were identical and belonged to the European lineage. However, from phylogenetic analysis, both strains appeared to be more closely related to viruses isolated between 1989 and 1995 than to viruses isolated more recently in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. The importance of expanding current equine influenza surveillance efforts is emphasised.

Virologic investigations of free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland.

We conducted virologic investigations on postmortem specimens from 261 free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland collected between 1990 and 2000. Fifty-four of 94 males had balanoposthitis; none of the 167 female bison examined had reproductive tract lesions. Peripheral blood, swabs, and various tissues were analyzed for bovine viruses as well as for viral DNA by bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 4 (BoHV-4) specific polymerase chain reaction (PCR) technique. An infectious bovine rhinotracheitis like BoHV-1 strain was isolated from the spleen of a female bison calf and additionally was detected by nested PCR from splenic tissue. None of the bison had significant antibody titers against BoHV-1, bovine herpesvirus 2, BoHV-4, caprine herpesvirus 1, cervid herpesvirus 1, or bovine viral diarrhea (BVD) virus-1. However, low antibody titers in two animals indicate that this European bison population has been exposed to BVD virus or BVD-like viruses and BoHV-2.

[Transmissible spongiform encephalopathies (TSE): old diseases with new explosive force]. / Transmissible Spongiforme Enzephalopathien (TSE): Alte Krankheiten mit neuer Brisanz.

In view of the first 64 BSE cases (date: 11.5.01) in German cattle herds an overview on TSE and their similarities and differences regarding clinic, pathogenesis and pathology is given. The mechanism of the unconventional agent, an infectious protein (prion), is explained based on the prion model of Stanley Prusiner. The knowledge on transmission, incubation time, host specificity as well as resistance and immunity drawn from experimentally infected animals is discussed. Thus, after oral infection prions are transported by lymphocytes from the stomach-intestinal tract to the spleen. The way to the CNS is still unknown. The presumption for crossing the species barrier is twofold: first the prions of different species have to be biochemically homologous and a genetical disposition has to exist. This is the case for BSE and the new variant of Creutzfeldt-Jakob-Disease (vCJD). There is evidence that in Great Britain so far 97 (date: 30.3.01) young people acquired vCJD due to consumption of food that contained bovine risk material. Regarding the infectious prion dosis brain, spinal cord and lymphoid tissues are regarded to be most dangerous. The principle of the BSE-test, its evidence as well as steps for prevention and control of BSE are presented.

Latency-associated transcripts of equine herpesvirus type 4 in trigeminal ganglia of naturally infected horses.

Equine herpesvirus type 4 (EHV-4) is a major respiratory pathogen of horses. Unlike most other members of the Alphaherpesvirinae, EHV-4 was regarded as non-neurotropic. Here, neural and lymphoid tissues of 17 horses have been analysed post-mortem. EHV-4 DNA was detected in 11 cases (65%) by PCR, exclusively in the trigeminal ganglia. In order to define the transcriptional activity, RNA preparations of 10 EHV-4 DNA-positive ganglia were investigated by nested RT-PCR. EHV-4-specific transcripts derived from genes 63 [herpes simplex virus type 1 (HSV-1) ICPO gene homologue] and 64 (HSV-1 ICP4 gene homologue) were detected in six trigeminal ganglia. In one other case, only gene 64-specific transcripts were present. All of the transcripts proved to be antisense orientated when a strand-specific RT-PCR was applied. Type-specific primers for gene 33 (encoding glycoprotein B) served to detect transcripts of an acute EHV-4-infection, which were found in only one of the six ganglia positive for gene 63- and gene 64-specific transcripts. Overall, these studies clearly demonstrate that EHV-4 is latent in trigeminal ganglia.