Two Different Macaviruses, ovine herpesvirus-2 and caprine herpesvirus-2, behave differently in water buffaloes than in cattle or in their respective reservoir species.

The ongoing global spread of "exotic" farm animals, such as water buffaloes, which carry their native sets of viruses, may bear unknown risks for the animals, into whose ecological niches the former are introduced and vice versa. Here, we report on the occurrence of malignant catarrhal fever (MCF) on Swiss farms, where "exotic" water buffaloes were kept together with "native" animals, i.e. cattle, sheep, and goats. In the first farm with 56 water buffaloes, eight cases of MCF due to ovine herpesvirus-2 (OvHV-2) were noted, whereas additional ten water buffaloes were subclinically infected with either OvHV-2 or caprine herpesvirus-2 (CpHV-2). On the second farm, 13 water buffaloes were infected with CpHV-2 and two of those succumbed to MCF. In neither farm, any of the two viruses were detected in cattle, but the Macaviruses were present at high prevalence among their original host species, sheep and goats, respectively. On the third farm, sheep were kept well separated from water buffaloes and OvHV-2 was not transmitted to the buffaloes, despite of high prevalence of the virus among the sheep. Macavirus DNA was frequently detected in the nasal secretions of virus-positive animals and in one instance OvHV-2 was transmitted vertically to an unborn water buffalo calf. Thus, water buffaloes seem to be more susceptible than cattle to infection with either Macavirus; however, MCF did not develop as frequently. Therefore, water buffaloes seem to represent an interesting intermediate-type host for Macaviruses. Consequently, water buffaloes in their native, tropic environments may be vulnerable and endangered to viruses that originate from seemingly healthy, imported sheep and goats.

Stimulation with a class A CpG oligonucleotide enhances resistance to infection with feline viruses from five different families.

Domestic cats are commonly affected by viral pathogens that induce lengthy infections with fatal outcomes. Prevention of viral propagation is of primordial importance in shelters and catteries, where cats from different backgrounds have narrow contacts. Oligonucleotides (ODN) containing cytosine-phosphate-guanosine motifs of class A (CpG-A) are highly potent synthetic inducers of innate antiviral mechanisms. The aim of this study was to test their ability to modulate innate immune responses and prevent viral replication as stand-alone agents in the domestic cat. CpG-A stimulation of feline peripheral blood mononuclear cells (PBMCs) enhanced their proliferation, increased the presence of co-stimulatory molecules on their surface and influenced their gene expression profiles in an antiviral orientation. Incubation of the supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial and fibroblastic origin induced expression of the antiviral myxovirus resistance (Mx) gene in these target cells, which also showed enhanced resistance to feline viruses from five distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. Most importantly, subcutaneous administration of CpG-A in domestic cats systemically increased the expression of Mx, reaching maximal levels within 24 h. Plasma from treated cats could furthermore inhibit viral replication in vitro. Altogether, our data highlight the promising potential of CpG-A to induce a preventive antiviral state in the cat and to protect feline populations against a broad range of virus infections.

A possible case of caprine-associated malignant catarrhal fever in a domestic water buffalo (Bubalus bubalis) in Switzerland.

BACKGROUND: Malignant catarrhal fever (MCF) is a fatal herpesvirus infection, affecting various wild and domestic ruminants all over the world. Water buffaloes were reported to be particularly susceptible for the ovine herpesvirus-2 (OvHV-2) causing the sheep-associated form of MCF (SA-MCF). This report describes the first case of possibly caprine-associated malignant catarrhal fever symptoms in a domestic water buffalo in Switzerland. CASE PRESENTATION: The buffalo cow presented with persistent fever, dyspnoea, nasal bleeding and haematuria. Despite symptomatic therapy, the buffalo died and was submitted to post mortem examination. Major findings were an abomasal ulceration, a mild haemorrhagic cystitis and multifocal haemorrhages on the epicardium and on serosal and mucosal surfaces. Eyes and oral cavity were not affected. Histopathology revealed a mild to moderate lymphohistiocytic vasculitis limited to the brain and the urinary bladder. Although these findings are typical for MCF, OvHV-2 DNA was not detected in peripheral blood lymphocytes or in paraffin-embedded brain, using an OvHV-2 specific real time PCR. With the aid of a panherpesvirus PCR, a caprine herpesvirus-2 (CpHV-2) sequence could be amplified from both samples. CONCLUSIONS: To our knowledge, this is the first report of malignant catarrhal fever in the subfamily Bovinae, where the presence of CpHV-2 could be demonstrated. The etiological context has yet to be evaluated.

Using scenario tree modelling for targeted herd sampling to substantiate freedom from disease.

BACKGROUND: In order to optimise the cost-effectiveness of active surveillance to substantiate freedom from disease, a new approach using targeted sampling of farms was developed and applied on the example of infectious bovine rhinotracheitis (IBR) and enzootic bovine leucosis (EBL) in Switzerland. Relevant risk factors (RF) for the introduction of IBR and EBL into Swiss cattle farms were identified and their relative risks defined based on literature review and expert opinions. A quantitative model based on the scenario tree method was subsequently used to calculate the required sample size of a targeted sampling approach (TS) for a given sensitivity. We compared the sample size with that of a stratified random sample (sRS) with regard to efficiency. RESULTS: The required sample sizes to substantiate disease freedom were 1,241 farms for IBR and 1,750 farms for EBL to detect 0.2% herd prevalence with 99% sensitivity. Using conventional sRS, the required sample sizes were 2,259 farms for IBR and 2,243 for EBL. Considering the additional administrative expenses required for the planning of TS, the risk-based approach was still more cost-effective than a sRS (40% reduction on the full survey costs for IBR and 8% for EBL) due to the considerable reduction in sample size. CONCLUSIONS: As the model depends on RF selected through literature review and was parameterised with values estimated by experts, it is subject to some degree of uncertainty. Nevertheless, this approach provides the veterinary authorities with a promising tool for future cost-effective sampling designs.

Epizootiologic investigations of selected abortive agents in free-ranging Alpine ibex (Capra ibex ibex) in Switzerland.

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems due to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

Causes of mortality and diseases in farmed deer in Switzerland.

To investigate diseases and causes of mortality in Swiss farmed deer, deer found dead or shot due to diseased condition between March 2003 and December 2004 were requested for a complete postmortem examination. One hundred and sixty-two animals were submitted. Perinatal mortality, necrobacillosis in 3 week to 6 month old deer, and endoparasitosis in 6 month to 2 year old deer were identified as the most important causes of loss, followed by ruminal acidosis, which was diagnosed in 22% of deer older than 1 year. Congenital malformations were observed in 15% of deer less than 6 months old. Reportable infectious diseases known as major problems in deer farming in other countries were rare (yersiniosis, malignant catarrhal fever) or not observed (tuberculosis, chronic wasting disease). Overall, the results indicate that the Swiss deer population does not present major health problems of concern for domestic animals.

The 2005/2006 avian influenza monitoring of wild birds and commercial poultry in Switzerland.

In October 2005, the second Swiss national avian influenza monitoring in wild waterfowl and commercial poultry with free range management started. Cloacal swabs were examined by real-time reverse transcription-polymerase chain reaction for both M gene of influenza A virus and H5 subtype. The monitoring (more than 2000 samples tested) documented the introduction of H5N1 in Swiss wild waterfowl in mid-February 2006. Until the end of March, 29 water bird carcasses were found H5 positive. In the same period, domestic poultry flocks with a permit of free-range management were kept under surveillance, with negative results.

Floppy kid syndrome caused by D-lactic acidosis in goat kids.

BACKGROUND: Goat kids with floppy kid syndrome have metabolic acidosis, muscle weakness, and depression but no dehydration. HYPOTHESIS: D-Lactate is the major component of acidemia in goat kids with floppy kid syndrome. ANIMALS: Fifty-five goat kids with floppy kid syndrome (group F) and 35 clinically healthy goat kids (group C). METHODS: Clinical, biochemical, microbiologic, virologic, parasitologic, and pathologic examinations. RESULTS: The animals in group F had a blood pH of 7.13 +/- 0.11 and a base excess of -17.8 +/- 3.8 mM, which were both lower than the values in the control animals (pH, 7.32 +/- 0.31; base excess, -0.1 +/- 2.7 mM; P < .001). Floppy kids had a significantly larger anion gap than healthy kids (31.2 +/- 3.7 versus 21.5 +/- 8.5 mM; P < .001). The concentration of L-lactate was lower in floppy kids than in healthy kids (0.67 +/- 0.49 versus 1.60 +/- 1.02 mM), but the concentration of D-lactate was higher in floppy kids (7.43 +/- 2.71 versus 0.26 +/- 0.24 mM; P < .001). Intravenous and oral administration of sodium bicarbonate in floppy kids resulted in a significant increase in blood pH and base excess and a decrease in the anion gap (P < .001). In addition, the concentration of L-lactate increased (P = .039). CONCLUSIONS AND CLINICAL IMPORTANCE: Metabolic acidosis in goat kids with floppy kid syndrome is caused by an increase in the plasma concentration of D-lactate.

Pro and contra IBR-eradication.

Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections such as infectious rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV, balanoposthitis (IBP), and abortion. Despite of a pronounced immune response, the virus is never eliminated from an infected host but establishes life-long latency and may be reactivated at intervals. Europe has a long history of fighting against BoHV-1 infections, yet, only a small number of countries has achieved IBR-eradication. Therefore, it seemed appropriate to review the reasoning pro and contra such a task. Clearly, the goal can indeed be achieved as has been demonstrated by a number of European countries. However, detection and stamping out of seemingly healthy virus carriers is inevitable in the process. Unfortunately, the use of vaccines is only of temporary and limited value. Therefore, there are numerous considerations to be put forward against such plans, including the high costs, the great risks, and the unsatisfactory quality of tools. If either control or eradication of IBR is nonetheless a goal, then better vaccines are needed as well as better companion tests. Moreover, better tools for the characterization of viral isolates are required. Collaborative actions to gather viral strains from as many countries as possible for inclusion into a newly created clustering library would be most advantageous.

Impairment of nuclear pores in bovine herpesvirus 1-infected MDBK cells.

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.

Concurrent infections with vector-borne pathogens associated with fatal hemolytic anemia in a cattle herd in Switzerland.

Bovine anaplasmosis is a vector-borne disease that results in substantial economic losses in other parts of the world but so far not in northern Europe. In August 2002, a fatal disease outbreak was reported in a large dairy herd in the Swiss canton of Grisons. Diseased animals experienced fever, anorexia, agalactia, and depression. Anemia, ectoparasite infestation, and, occasionally, hemoglobinuria were observed. To determine the roles of vector-borne pathogens and to characterize the disease, blood samples were collected from all 286 animals: 50% of the cows were anemic. Upon microscopic examination of red blood cells, Anaplasma marginale inclusion bodies were found in 47% of the cows. The infection was confirmed serologically and by molecular methods. Interestingly, we also found evidence of infections with Anaplasma phagocytophilum, large Babesia and Theileria spp., and Mycoplasma wenyonii. The last two species had not previously been described in Switzerland. Anemia was significantly associated with the presence of the infectious agents detected, with the exception of A. phagocytophilum. Remarkably, concurrent infections with up to five infectious vector-borne agents were detected in 90% of the ill animals tested by PCR. We concluded that A. marginale was the major cause of the hemolytic anemia, while coinfections with other agents exacerbated the disease. This was the first severe disease outbreak associated with concurrent infections with vector-borne pathogens in alpine Switzerland; it was presumably curtailed by culling of the entire herd. It remains to be seen whether similar disease outbreaks will have to be anticipated in northern Europe in the future.

[Import risk analysis in animal disease control]. / Importrisikoanalysen in der Tierseuchenbekämpfung.

At the Swiss Federal Veterinary Office risk analyses are conducted according to international standards. A risk analysis contains the elements risk management, risk assessment and risk communication. A risk assessment is based on risk profile, hazard identification and a pathway model. All available information is gathered, documented and assessed and the risk estimated. The question. "What is the probability that unprocessed wild boar meat imported to Switzerland from the federal state Mecklenburg Western Pommerania is contaminated with classical swine fever virus?" was answered by a release assessment. The hazard identification recognized classical swine fever virus and attenuated live virus vaccine used for oral immunization as hazards. The probability of contamination was estimated to be small. The question: "What is the likelihood to introduce Aujeszky's disease to Switzerland and infect the indigenous pig population with the disease, by means of importing pork and meat products?" was answered by assessing the release, exposure and resulting consequences. The risk of an infection of the indigenous pig population was estimated to be very small, as 80% of the imported products derive from countries or zones free from Aujeszky's disease. Furthermore the majority of the imported products are processed. The strict implementation of the regulations governing feeding of food wastes to pigs reduces the probability of exposure. In all assessments the risk management decides on a strategy to deal with the risk, taking into consideration the results and recommendations derived from the risk assessment as well as other relevant factors.

Both viral and host factors contribute to neurovirulence of bovine herpesviruses 1 and 5 in interferon receptor-deficient mice.

Herpes simplex virus (HSV) type 1 and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5) can use the same cellular receptor for entry, but only HSV is known to cause disease in mice. We hypothesized that components of either the innate or the adaptive immune system, or a combination of both, were responsible for curbing replication of BHVs in mice. Therefore, wild-type mice as well as mice with various combined genetic deficiencies in the alpha/beta interferon receptor or gamma interferon receptor and in the ability to produce mature B and T lymphocytes (RAG-2 deletion) were infected with BHV-1 and BHV-5 and monitored clinically, serologically, histopathologically, and virologically. A functional immune system protected the mice from disease and death due to BHV infection, and the immune response was Th1 like. BHV-5 was transported to the central nervous system by the axonal pathway, whereas viremia was required for this outcome with BHV-1. The alpha/beta interferon system was able to obstruct quantitative spread of the viruses in the infected organism. The gamma interferon system had a protective effect against BHV-1, even in mice with the RAG-2 deletion. In contrast, the same mice succumbed to neurological disease and death upon infection with BHV-5. Productively infected neurons were detected only in BHV-5-infected mice with an intact gamma interferon system. We conclude that the alpha/beta interferon system had a protective effect, while an intact gamma interferon system was required for efficient replication of BHV-5 in mouse neurons and for the development of neurological disease.

The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification.

Chemical modification of the proteins bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, alpha-lactalbumin, beta-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.

Identification and quantification of ovine gammaherpesvirus 2 DNA in fresh and stored tissues of pigs with symptoms of porcine malignant catarrhal fever.

Cases of porcine malignant catarrhal fever were analyzed by a combination of identification and quantitation of ovine gammaherpesvirus 2 DNA in a variety of paraffin-embedded tissues from diseased pigs, serology, and exclusion of primary porcine gammaherpesviruses. In spite of reduced signal due to fixation and paraffin embedding, ovine gammaherpesvirus 2 DNA in pig brains exceeded the amounts found in sheep brains by orders of magnitude.

Herpesviruses: balance in power and powers imbalanced.

The first veterinary herpesvirus symposium, organized under the patronage of the European Society for Veterinary Virology (ESVV) and the Swiss Societies for Microbiology (SGM-SSM), was held at the University of Zurich, Switzerland, on 22nd and 23rd of March 2001. The congress was divided into six sessions. The first session was dedicated to introductory lectures towards the main topics of the symposium, namely pathogenesis, immune response, and gene therapy. Session 2 was committed to new insights into herpesvirus-related gene therapy and vaccination. Specific and general aspects of the immune response against herpesviruses were presented in session 3, while session 4 was dedicated to virus replication. Session 5 was dedicated to a variety of poster presentations. Finally, session 6 revealed new insight into the pathogenesis of different herpesviruses. The present article summarizes the contributions and draws a new view of the herpesviruses. The herpesviruses have apparently found a multi-dimensionally balanced position between the powers of "cytopathogenicity" and "tumorigenicity" on one hand and "immunogenicity" and "tolerogenicity" on the other hand. As long as the different powers stay in balance, no or little clinical disease may be expected in association with herpesvirus infections. However, unbalanced actions of those powers may lead to disease.

The significance of the Golgi complex in envelopment of bovine herpesvirus 1 (BHV-1) as revealed by cryobased electron microscopy.

Nucleocapsids of herpesviruses originate in the nucleus of host cells and bud through the inner nuclear membrane acquiring tegument and envelope. The release of the enveloped virus particle from the perinuclear space is unknown. Cryobased electron microscopic imaging revealed enveloped virus particles within cisterns associated with the perinuclear space, a pre-Golgi compartment connecting Golgi cisterns to the perinuclear space, and enveloped virus particles in Golgi cisterns where they are packaged into transport vacuoles by membrane fission. To our knowledge, our images show for the first time the connectivity from the perinuclear space to Golgi cisterns. The data strongly indicate an intracisternal transport of enveloped virus particles from the budding site to the packaging site. Budding starts by condensation at the inner membrane. Condensation involving the viral envelope and peripheral tegument was persistent in virus particles within perinuclear space and associated cisterns. Virus particles within Golgi cisterns and transport vacuoles originating by Golgi membrane fission, however, lacked condensation. Instead, spikes were clearly evident. The phenomenon of condensation is considered likely to be responsible for preventing fusion of the viral envelope with cisternal membranes and/or for driving virions from the perinuclear space to Golgi cisterns. Glycoprotein K is discussed to likely play a role in the intracisternal transportation of virions. In addition to the pathway including intracisternal transport and packaging, there were clear indications for the well-known pathway involving wrapping of cytoplasmic nucleocapsids by Golgi membranes. The origin of the cytoplasmic nucleocapsids, however, remains obscure. Lack of evidence for release of nucleocapsids at the outer nuclear membrane suggests that the process is very rapid, or that nucleocapsids pass the nucleocytoplasmic barrier via an alternative route.