László Egyed

Seasonal activity and tick-borne pathogen infection rates of Ixodes ricinus ticks in Hungary

Ixodes ricinus is the most important tick species in Europe as it is most widely distributed and transmits the majority of tick-borne zoonotic pathogens. As limited data are available for Hungary, the aim of the present study was to investigate the seasonal timing of questing by I. ricinus and the infection rate of this tick species with all major tick-borne zoonotic pathogens. Monthly collections of I. ricinus were carried out over 3 consecutive years by dragging a blanket in 6 biotopes representing different areas of Hungary. Altogether, 1800 nymphs (300 per collection point) were screened as pooled samples (each of 5 specimens) by PCR-based methods for tick-borne pathogens. I. ricinus larvae, nymphs, and adults had bimodal activity patterns with a major peak in the spring. As newly moulted ticks of all stages are thought to emerge in the autumn of each year, it appears that most newly emerged ticks delayed their questing until the following spring. The minimum prevalence of Borrelia burgdorferi sensu lato was 2.5%. Borr. afzelii, Borr. burgdorferi sensu stricto, Borr. garinii, Borr. lusitaniae, and Borr. valaisiana were identified by hybridization. The minimum infection rate with spotted fever group rickettsiae was 1.9%. Rickettsia helvetica was identified in all biotopes. The minimum prevalence of Anaplasma phagocytophilum, Babesia divergens and Bab. microti was low (0.3-0.5%). Bartonella spp.-, Francisella tularensis-, and TBE virus-specific amplification products were not detected. Relative to the results of comparable studies carried out in the Carpathian Basin, the prevalence of tick-borne pathogens was low in Hungary. This might be attributed to the climatic difference between the lowland areas of Hungary and submountain areas of the surrounding countries involved in the studies.

Bovine kobuvirus in Europe.

To the Editor: Picornaviruses (family Picornaviridae) are small, nonenveloped viruses with single-stranded, positive-sense genomic RNA. Picornaviruses are currently divided into 8 genera: Enterovirus, Aphthovirus, Cardiovirus, Hepatovirus, Parechovirus, Erbovirus, Teschovirus, and Kobuvirus (1). To date, the genus Kobuvirus consists of 2 officially recognized species, Aichi virus and Bovine kobuvirus, and 1 porcine kobuvirus as a candidate species (2–4). Aichi virus (strain A846/88) was first isolated in 1991 from feces of a person with acute gastroenteritis (2). Bovine kobuvirus (strain U-1) was detected in 2003 in bovine serum and fecal samples from clinically healthy cattle (3); in 2008, it was isolated from cattle with diarrhea (5). Aichi virus and bovine kobuvirus were first isolated in Japan. Porcine kobuvirus (strain S-1-HUN) was recently identified from domestic pigs in Hungary (4). Aichi viruses have been also detected in other countries in Asia (6), Europe (7,8), South America (7), and northern Africa (9). Bovine kobuvirus, however, has not been detected outside Asia (Japan and Thailand) (3,5).

Molecular evidence of Rickettsia helvetica and R. monacensis infections in Ixodes ricinus from Hungary

The genus Rickettsia consists of strictly intracellular bacteria that fall into one of three groups: the typhus group, the scrubtyphus group, or the spotted-fever group. Some European ticks are know to harbour rickettsiae of the spotted-fever group, such as Ri. conorii, Ri. massiliae, Ri. rhipicephali, Ri. slovaca, Ri. aeschlimannii, Ri. helvetica and Ri. monacensis (Beati et al., 1996; Raoult and Roux, 1997; Fournier et al., 2000; Sekeyova et al., 2000; Parola and Raoult, 2001a, b; Beninati et al., 2002; Simser et al., 2002; Fernández-Soto et al., 2003; Fournier et al., 2003; Parola, 2004). In Europe, Ri. aeschlimannii, Ri. conorii and Ri. rhipicephali appear to be transmitted only by Hyalomma and Rhipicephalus spp. and their distribution is therefore probably restricted to the warmer south (Parola and Raoult, 2001a, b; Fernández-Soto et al., 2003; Parola, 2004). In contrast, Ri. slovaca (transmitted by Dermacentor spp.) and Ri. massilisae (transmitted by Dermacentor or Hyalomma spp.) probably occur throughout Europe (Rydkina et al., 1999; Lakos, 2002; Raoult et al., 2002; Márquez et al., 2003). Although Ri. helvetica was only described as a species in 1993 (Beati et al., 1993), it has already been reported from several countries in southern, western, central and northern Europe over the last decade. It is transmitted mainly by Ixodes spp. (Christova et al., 2003; Parola, 2004) and has been detected, at prevalences varying from 1%– 16%, in ‘anthropophilic’ I. ricinus collected in Europe (Beati et al., 1994; Nilsson et al., 1997; Parola et al., 1998; Avsic-Zupanc et al., 1999; Bacellar, 1999; Nilsson et al., 1999a; Beninati et al., 2002; Christova et al., 2003; Sanogo et al., 2003; Fernández-Soto et al., 2004; Hartelt et al., 2004; Nielsen et al., 2004). Over the last 6 years, several cases of human infection with Ri. helvetica have been detected in Europe — in France, Italy, Switzerland and Sweden (Nilsson et al., 1999a, 2002; Baumann et al., 2003; Fournier et al., 2004; Parola, 2004) — and high seroprevalences (9%–12%) of human infection have been observed among highrisk groups in France, Switzerland and Denmark (Fournier et al., 2000; Baumann et al., 2003; Nielsen et al., 2004). Although acute human infection with Ri. helvetica is generally associated with relatively mild symptoms, such as headache, fever, myalgia and arthralgia (Fournier et al., 2004), it has been suggested that these rickettsiae may also contribute to a chronic granulomatous process, as seen in sarcoidosis, and to chronic perimyocarditis, which may result in sudden death (Nilsson et al., 1999b, 2002, 2005). Nevertheless, the possible association between Ri. helvetica and serious human disease remains a matter of controversy (Walker, 2003; Planck et al., 2004). Recently, a ‘new’Rickettsia sp. was detected in I. ricinus in Spain, Germany, Italy, Slovakia, Albania, Bulgaria and Japan, and named Ri. monacensis (Sekeyova et al., 2000; Simser et al., 2002; Beninati et al., 2002; Ishikura et al., 2002; Christova et al., 2003; Sanogo et al., 2003; FernándezSoto et al., 2004). In terms of their 16S ribosomal DNA (rDNA), gltA, ompA and 17-kDa gene sequences, all the strains of Ri. monacensis that have been investigated (such as IrR/Munich, IRS3, IRS4, IrITA2 IrITA3 and In56) form a separate cluster within the spotted-fever group (Sekeyova et al., 2000; Beninati et al., 2002; Ishikura Annals of Tropical Medicine & Parasitology, Vol. 99, No. 3, 325–330 (2005)

Experimental infection of goats with tick-borne encephalitis virus and the possibilities to prevent virus transmission by raw goat milk

Objectives: The aim of this work was to study the tick-borne encephalitis virus (TBEV) infection of goats and the possibilities to prevent human milk-borne infections either by immunizing animals or the heat treatment of milk. Methods: An experiment was conducted with 20 milking goats. Ten goats (half of them immunized) were challenged with live TBEV and 10 were left uninfected. Clinical signs and body temperatures of the animals were recorded and milk samples were collected daily. The presence of viral RNA and infectious virions in milk were detected by RT-PCR and intracerebral inoculation of suckling mice, respectively. Milk samples containing infectious virions were subjected to various heat treatment conditions and retested afterwards to assess the effect on infectivity. Results: The infected goats did not show any clinical signs or fever compared to uninfected ones. Infectious virions were detected for 8–19 days from the milk samples (genome for 3–18 days by PCR) of infected goats. Immunized goats did not shed the virus. After heat treatment of the milk, the inoculated mice survived. Conclusions: Goats shed the virus with their milk without showing any symptoms. Human milk-borne infections can be avoided both by immunizing goats and boiling/pasteurizing infected milk.

Histological studies of bovine herpesvirus type 4 infection in non-ruminant species.

The pathology of bovine herpesvirus type 4 (BHV-4) infection was studied in cats, rabbits and guinea pigs. Twenty kittens, twenty-two rabbits and ten guinea pigs, some treated with glucocorticoid-were inoculated with a BHV-4 strain of feline origin, via various routes of inoculation (conjunctival, intranasal, peritoneal). Clinical signs were recorded. After euthanizing at different post inoculation days macro- and microscopic changes were observed by necropsy and in hematoxylin-eosin stained histological sections. The presence of the virus in organs was detected by immunohistochemistry and a nested PCR assay. Inclusion bodies and monoclonal antibody-stained cells were found in the conjunctiva, trachea, lungs, spleen and lymph nodes. Most of the lesions were localized to the respiratory and the immune system. The macro- and microscopic lesions and clinical signs were more severe in kittens and guinea pigs. The histological data indicated that cats, especially kittens, were susceptible for BHV-4 and the infection was not confined to the urinary bladder.

Detection of genotype 1 and 2 bovine noroviruses in Hungary.

CALICIVIRUSES (family Caliciviridae) are small, non-enveloped viruses with single-stranded, positive-sense genomic RNA that are classified into four genera: Vesivirus, Lagovirus, Norovirus and Sapovirus ([Mayo 2002][1]). Norwalk and Sapporo viruses are prototype strains of the Norovirus and

Babesia microti infection of anthropophilic ticks (Ixodes ricinus) in Hungary

(2003). Babesia microti infection of anthropophilic ticks (Ixodes ricinus) in Hungary. Annals of Tropical Medicine & Parasitology: Vol. 97, No. 3, pp. 317-319.

Symptomless intrauterine transmission of bovine herpesvirus 4 to bovine fetuses.

Blood samples of 31 healthy calves and their dams taken immediately after calving before colostrum uptake, and at days 11, 23 and 8 weeks, spleens of seven stillborn calves were analysed in order to determine the source and time of bovine herpesvirus type 4 infection. All the calves were born as seronegatives, while all cattle were seropositives. Viral DNA were amplified by a nested PCR assay from 54.8% of peripheral blood leukocyte samples of newborn calves taken before colostrum uptake, and from all cattle and from their colostrums. Real time PCR detected higher virus level in peripheral blood leukocytes in adult cattle, then in their newborn calves. Bovine semen cells (spermatozoa and leukocyte fractions), spleens of stillborn calves also carried viral genomes. Our results prove, that bovine fetuses can be infected in utero by BoHV-4, but are born as seronegatives. After human examples this is the first report in veterinary virology on intrauterine transmission of a herpesvirus without acute consequences. This phenomenon could explain the low antigenicity of BoHV-4 proteins and lack of neutralizating antibodies. BoHV-4, a gammaherpesvirus, could serve as an animal model for studying inapparent herpesviral infections of human fetuses.

PCR DETECTION OF BOVINE HERPESVIRUSES FROM NONBOVINE RUMINANTS IN HUNGARY

Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, −2, and −4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank.

Tick-Borne Encephalitis and Lyme Disease in Hungary: The Epidemiological Situation Between 1998 and 2008

Diagnosed cases of tick-borne encephalitis (TBE) and Lyme disease (LD) have been reportable infectious diseases in Hungary since 1977 and 1998, respectively. Clinically diagnosed cases have been registered in the National Database of Epidemiological Surveillance System (NDESS). All reported TBE cases are confirmed by laboratory serological and, if necessary, PCR tests, whereas the registered cases of LD are mainly based on the appearance of erythema migrans concurring with possible exposure of tick bite. Our work is the first comparative epidemiological and geographical information analysis of these 2 diseases together. The following demographic data from each individual case (703 TBE and 13,606 LD) recorded in the NDESS were used: Sex, age, the starting date and place of the onset of disease, and a short report from the affected person. The descriptive epidemiological analysis of incidence was carried out using directly standardized rates, and smoothed indirectly standardized incidence ratios were calculated by hierarchical Bayesian methods at the municipality level using a Rapid Inquiry Facility (RIF). The average yearly incidence rate of TBE was 0.64 per 100,000 inhabitants (range, 0.46-0.84) and of LD was 12.37 per 100,000 inhabitants (range, 9.9-18.1), with the highest incidence rates in 1998 for TBE and 2008 for LD. The most affected age groups were men between 15 and 59 years of age for TBE, and women between 45 and 64 years of age for LD. Seasonality, based on the starting date of the illness, was also characterized. Extended areas of high risk were identified in western and northern Hungary, illustrated on high-resolution (municipality level) maps. On the basis of our analysis, it is possible to associate areas and periods of high-risk with characteristics (sex, age, residence) of groups most affected by tick-borne diseases in Hungary.