Difference in susceptibility of small rodent host species to infestation by Ixodes ricinus larvae.

In a laboratory experiment (repeated eight times) individuals of six small rodent species were mixed with 500 Ixodes ricinus larvae for 24 h, than over a 3-day-long observation period engorged ticks fallen from individually kept rodents were counted. Forty engorged larvae fell from the hosts, 25% from striped field mice, 32.5% from house mice, 22.5% from common voles, 10% from bank voles, while wood mouse and steppe mouse proved to be almost free of successfully infesting ticks (both 5%). Statistical analysis indicated, that tick burden differed significantly among host species. Similarly significant differences were shown between sensitivity of two related species, house mouse and steppe mouse, and more ticks were collected on the second observation day compared to the first day. The difference in susceptibility of hosts to Ixodes infestation could be due to various anti-tick behavior habits of hosts, or difference in preference of the ticks between host species.

A synopsis of records of myxozoan parasites (Cnidaria: Myxozoa) from shrews, with additional data on Soricimyxum fegati from common shrew Sorex araneus in Hungary and pygmy shrew Sorex minutus in Slovakia.

Myxozoans (Cnidaria: Myxozoa) are almost exclusively endoparasites of aquatic vertebrates and invertebrates, with the notable exception being two species of Soricimyxum Prunescu, Prunescu, Pucek et Lom, 2007 described from terrestrial shrews (Soricidae) in central Europe. Myxospores of the two parasites are morphologically indistinguishable, but have SSU rDNA sequences that differ by about 4%. Herein, we report additional molecular and histology data from Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 from common shrew (Sorex araneus Linnaeus) from Hungary, and add a new geographic record for S. fegati in pygmy shrew (Sorex minutus Linnaeus) from Slovakia. A limited survey of shrews from the northern United States, Blarina brevicauda Say and Sorex sp. from New York, and Sorex spp. from Oregon, did not discover any infections, which is in stark contrast to the relatively high infection rates (up to 66%) in European shrew populations. We also provide a summary and discussion of literature records of species of Soricimyxum and a host survey. Given the lack of distinguishing morphological or morphometric characters between Soricimyxum spp., and the overlap in vertebrate hosts and geographic ranges, unambiguous identification of these closely related shrew parasites can presently only be achieved through sequence comparison of one or more variable SSU rDNA regions.

Subclinical Tick-Borne Encephalitis Virus in Experimentally Infected Apodemus agrarius.

OBJECTIVES: In this study, we investigated the dose dependence of tick-borne encephalitis virus (TBEV) infection in one of the reservoirs, i.e. Apodemus agrarius, a small rodent species. METHODS: The animals were challenged with TBEV per os and intramuscularly with infectious doses ranging from 1 to 1,500 plaque-forming units (pfu). Clinical signs were recorded and clinical and pathological features were evaluated by histological, immunohistochemical, and serological methods. RESULTS: High perorally administered infectious doses resulted in virus replication in the brain, which is the first sign of subclinical viral encephalitis in the Apodemus genus. The animals seroconverted at infectious doses greater than 100 pfu, and all animals remained asymptomatic. CONCLUSION: Our work shows the first evidence that subclinical TBEV encephalitis may occur in Apodemus species, depending on the virus load of the inoculum. The antiviral response of the local innate immune system may influence the resistance of Apodemus individuals to lower infectious doses. Per oral/nasal infection seems to be more dangerous for the host than other routes of infection.

A novel myxozoan parasite of terrestrial mammals: description of Soricimyxum minuti sp. n. (Myxosporea) in pygmy shrew Sorex minutus from Hungary.

As part of a biodiversity study in northwestern Hungary, we conducted a parasitological survey of small mammals. In both common shrews (Sorex araneus Linnaeus) and pygmy shrews (Sorex minutus Linnaeus), we found myxospores of a species of Soricimyxum Prunescu, Prunescu, Pucek et Lom, 2007 (Myxosporea) and plasmodia in the bile ducts within the liver. Spores from both species of shrewswere morphologically and morphometrically indistinguishable, but differed in their SSU rRNA gene sequences by 3.3%. We identified spores and developmental stages from the common shrew as Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007, based on morphometric data and DNA sequence similarity. Spores from the pygmy shrew were only 96.7% similar to S. fegati, hence we identified them as a novel myxosporean Soricimyxum minuti sp. n. This is only the second myxosporean parasite species described from mammals.

Cultivable internal bacterial flora of ticks isolated in Hungary.

From six sampling sites in north-western Hungary 126 questing ticks of three species (Ixodes ricinus, Dermacentor reticulatus, Haemaphysalis concinna) were sampled. After inactivating the external bacteria on the surface of ticks, the internal bacterial flora was cultured (two kinds of agar media at three temperatures with aerobic and anaerobic conditions were applied), and 116 strains were isolated. Our results showed, that after a blood meal the bacterial contamination of ticks was much higher in Ixodes and Dermacentor, than in Haemaphysalis, indicating different host range or blood meal habits. Most (89.7 %) of the bacteria were Gram-positive, the most frequent genera were the Staphylococcus (18.1 %) and Bacillus (7.8 %). The percentage of bacteria part of the skin and mucosal flora was 21.6 %. Among the environmental bacteria 14 were found with reported medical importance. The results show, that members of some genera are able to replicate inside the ticks (Mycobacterium, Bacillus) which can increase their potential risk. Isolated bacteria/tick ratio continuously grew from larvae to adults, indicating that larvae probably are hatched sterile, but later bacterial uptake from the environment and from the hosts increases bacterial contamination. Ten anaerobic bacteria were cultured, mostly Propionibacterium acnes, a facultative skin pathogen. No significant differences were found between the isolated bacteria of the six sampling sites. Our work showed, that internal bacterial community of ticks is diverse, novel strains were isolated several with medical importance, some bacteria multiplicate inside ticks, and that ticks continuously take up bacteria from the environment. Our study first described anaerobic bacteria from ticks.

Monitoring the diel activity of Ixodes ricinus ticks in Hungary over three seasons.

Ixodes ricinus is the most common tick species in Europe and vector of many diseases of humans. The risk of contracting such a disease is influenced by many factors, but one of the crucial points is questing activity of unfed ticks. In order to supplement the few literature data on patterns of diel activity of this tick species and to examine the correlations between data on diel activity of ticks and their small mammal hosts and some meteorological variables, a survey was performed. Diel activity of questing I. ricinus and small rodents was studied in a known natural tick-borne encephalitis virus focus over 7 months at one sampling day monthly. 1,063 I. ricinus ticks and 25 rodents were sampled. Air temperature and humidity data were also recorded in the 24 study plots at time of sampling. From April to October questing activity of nymphs increased in the 3-h-period after sunrise, comparing to activity of the 3 h before sunrise. Proportion of nymphs sampled 3 h after sunset compared to total sampled nymphs 3 h before and 3 h after sunset showed correlation to activity of rodents. In the period of April-July both nymphs and larvae showed stronger activity from sunrise to sunset, this turned to dominant nighttime activity in August-September, whereas activity changed to equal in day and night in October. Our results indicate that natural light and rodent population have positive effect on questing activity of I. ricinus.

Features of Engorgement of Ixodes ricinus Ticks Infesting the Northern White-Breasted Hedgehog in an Urban Park.

In this work we exploited the parallel dense tick and hedgehog populations of an urban park in Budapest, Hungary as a good host-parasite model to obtain detailed data about this physiological relationship. Over a 27-week period from April to October, 57 hedgehogs were captured in an urban park and kept for 10-14 days in animal house. All dropped off ticks were sampled, which allowed us to draw more a detailed picture of Ixodes ricinus-hedgehog relationships. The results indicated that the hedgehog is an effective host for ticks (prevalence: 100%) and the mean intensity of infestation was 83.25. Of the male ticks, 68.42% dropped off dead; 1.56% of the dropped off nymphs and 11.4% of the larvae finished their bloodmeal with red cuticles, while 5.79% of the females could not finish their blood meal, and dropped off dried, dead, or shrunken. We applied novel statistical methods of survival analysis of prevalent cohorts to estimate the whole attachment times of ticks from the observed attachment times, having no information about when the ticks attached to their hosts. Mean attachment times were 4 days for larvae, 5 days for nymphs, 10 days for females, and 8 days for males. On the first day after capture of the hosts, fewer females, nymphs, and larvae detached engorged than had been predicted, but this was not true for males. Mean intensity of infestation per host was 1.4 for males, 6.7 for females, 45.0 for nymphs, and 29.3 for larvae. As regards seasonality, the activity of all stages of ticks consisted of several smaller peaks and considerably differed by season. Studies of the dense tick-host populations of this natural habitat could provide further valuable data about tick-host relations, the data of which cannot be drawn from most other hedgehog habitats.

Tick-borne encephalitis epidemic in Hungary 1951-2021: The story and lessons learned.

The authors analysed epidemiological data of the Hungarian tick-borne encephalitis epidemic from the past seven decades. A total of 911 meningitis serosa cases were described from 1930-1950 s by local hospital physicians, indicating that the virus had been present in the country decades before its official identification in 1952. The virus spread freely in the 1950s-1960s, occupying almost all habitats where ticks occurred in large numbers. The increasing number of cases drove authorities to classify this illness as a notifiable disease in 1977 and to organize the first measures to stop the epidemic. Statistical analysis revealed that the large-scale vaccination launched from the 1990s was responsible for the sharp decrease in the number of human cases from 1997. A significant negative correlation was found between the number of vaccine doses sold and human cases 6 years later. The TBEV endemic area covers 16.57% of the territory and 16.65% of the population of the country. In the last 10 years, 186,000 vaccine doses/year in average were enough to keep the incidence of human TBEV infections between 0.45 and 0.06/100,000 persons. A 20-year-long study found evidence for easing clinical signs in TBEV-infected hospitalized patients. Statistics found a sharp decrease in the number of samples sent for TBEV diagnosis after 1989. Male dominance of patients was characteristic of the epidemics since the 1940s, but now analysis of detailed data from the 1981-2021 period (60.5%-87.5%) proved the statistical significance of this dominance. Obviously, the voluntary vaccination programme was the tool which broke the spread of the epidemic. Widespread public awareness of the disease and the tick vector, probable evolutionary spread of less pathogenic virus strains supplemented with the vaccination campaign led to a negligible level of human TBE cases in Hungary in the last years.

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.

Molecular detection of novel astroviruses in wild and laboratory mice.

Pooled fecal specimens collected from striped field mice (Apodemus agrarius), yellow-necked mice (Apodemus flavicollis), and bank voles (Myodes glareolus) and individual stool samples collected from laboratory mice were tested for the presence of picornaviruses and astroviruses. Picornavirus RNA was detected only in one striped field mouse sample pool, while astrovirus RNA was detected in two yellow-necked mouse sample pools and in six of the 121 laboratory mouse samples. In a 234-amino acid (aa) fragment of the viral RNA dependent RNA polymerase (RdRp), the wild mouse picornavirus revealed the closest homology to the canyon mouse (Peromyscus crinitus) (93 % aa) and canine kobuviruses (92 % aa) and to Aichi virus (88 % aa). The two astroviruses detected in the yellow-necked mouse samples shared 77 % aa homology with each other in the partial (125 aa) RdRp region, 61-62 % aa homology with rat astroviruses and only 54-58 % aa homology with the house mouse (Mus musculus) astrovirus strain USA/2008/M52. The six laboratory mouse astroviruses displayed 97-100 % aa homology to each other, and shared 71-77 % aa homology with the yellow-necked mouse astroviruses, 58-59 % aa homology with rat astroviruses and 55-56 % aa homology with strain USA/2008/M52. The sequence of a 3,263 bp genome segment including the partial ORF1b (RdRp), complete ORF2 (capsid precursor), and 3' NTR of a research mouse astrovirus strain (TF18LM) was determined. The full-length ORF2 showed low identities (17-34 % aa) with other members of the Mamastrovirus genus and only 17 % aa homology with the house mouse astrovirus strain USA/2008/M52, indicating that AstVs described in this study represent a novel Mamastrovirus species. The relevance of astrovirus infection and its effect on biomedical research conducted in mice needs to be investigated.

Effect of low-pathogenicity influenza virus H3N8 infection on Mycoplasma gallisepticum infection of chickens.

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.

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.

Survival of Tick-Borne Encephalitis Virus in Goat Cheese and Milk.

Survival of tick-borne encephalitis virus was studied from pasteurized and unpasteurized goat milk and from salted/unsalted and spiced/unspiced cheese made from goat milk inoculated with low and high litres of infective virus. Both soft (63 °C, 30 min) and fast (72 °C, 15 s) pasteurization conditions destroyed viable virus particles. A small amount of infective virus could be detected only for 5‒10 days from milk, and from unsalted cheese. From milk inoculated with a higher amount of virus, infectious viral particles were detectable for 20‒25 days and from unsalted cheese samples for 10‒15 days, independently of the use of spices. Pasteurization and salt treatment made goat milk and cheese safely consumable. These two methods must be used when making any human food from goat milk to avoid milk-borne human TBEV infections.

Hungarian tick-borne encephalitis viruses isolated from a 0.5-ha focus are closely related to Finnish strains.

Four tick-borne encephalitis virus strains were isolated from a small 0.5-ha focus over a six-year-long period (2011-2016) in Hungary. Two strains with identical genomes were isolated from Ixodes ricinus and Haemaphysalis concinna two months apart, which shows that the virus had not evolved separately in these tick species. Whole-genome sequencing of the virus revealed that the isolates differed from each other in 4 amino acids and 9 nucleotides. The calculated substitution rates indicated that the speed of genome evolution differs from habitat to habitat, and continuously changes even within the same focus. The amino acid changes affected the capsid, envelope, NS2a and NS5 genes, and one mutation each occurred in the 5' and 3' NCR as well as the premembrane, NS2a and NS5 genes. Phylogenetic analyses based on complete coding ORF sequences showed that the isolates belong to the European subtype of the virus and are closely related to the Finnish Kumlinge strains, the Bavarian isolate Leila and two isolates of Russian origin, but more distantly related to viruses from the neighbouring Central European countries. These isolates obviously have a common origin and are probably connected by migrating birds. These are the first published complete Hungarian TBEV sequences.

Detection of bovine herpesvirus 4 DNA in aborted bovine fetuses.

The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.

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.

[Rickettsia helvetica: an emerging tick-borne pathogen in Hungary and Europe]. / Rickettsia helvetica: egy növekvo jelentoségu kullancs terjesztette kórokozó hazánkban es Európában.

Rickettsia helvetica belonging to spotted fever group rickettsiae was recently detected by polymerase chain reaction followed by sequencing in European sheep ticks (Ixodes ricinus) from Hungary. Current knowledge on these rickettsiae and the clinical and diagnostic aspects of R. helvetica infection is summarized. In acute cases, R. helvetica is generally responsible for flu-like symptoms. Nevertheless, recent data indicate that in chronic cases, these rickettsiae can be responsible for perimyocarditis resulting sudden cardiac death and might play a role in the pathogenesis of aortic valve disease. The diagnosis can be based on serological, molecular and histological methods. A summary of the information available from Hungary and neighbouring countries on the prevalence of tick-borne encephalitis virus, Anaplasma, Borrelia, Francisella, Rickettsia and Babesia infections in I. ricinus is also presented.

[Babesia microti and Anaplasma phagocytophilum: two emerging zoonotic pathogens in Europe and Hungary]. / Babesia microti és Anaplasma phagocytophilum: két növekvo jelentoségu kórokozó Európában és hazánkban.

Babesia microti and Anaplasma phagocytophilum was recently reported with a minimum prevalence of 0.9 and 1.3% in Hungary based on the PCR-sequencing analysis of 452 European sheep ticks (Ixodes ricinus). These results and the epidemiological data of the neighbouring countries indicate that human cases caused by these pathogens may occur in the country. The aim of the present paper is to summarise the current knowledge on the morphology, life cycle and distribution of B. microti and A. phagocytophilum, and the epidemiology, clinical features, diagnosis, treatment and control of babesiosis and granulocytic anaplasmosis.

Bank voles show high seropositivity rates in a natural TBEV focus in Hungary.

Rodents captured in a known tick-borne encephalitis virus (TBEV) focus were serologically surveyed for 4 years, with 28 visits. The collected sera were analysed by virus neutralization test. Bank vole (Myodes glareolus) had a significantly higher incidence rate of antibodies to TBEV (20.5%) than Apodemus flavicollis (3.7%) and Apodemus agrarius (4.6%). In all species, rates were higher in adults (6.8%) than in juveniles (1.7%). A higher incidence rate was observed in female A. flavicollis individuals (6.7%) than in males (1.5%). Smaller bank vole population coincided with lower (1.2-4.8%) seropositivity in all small rodents, while more abundant bank vole population meant higher (17.9%) total seropositivity. The TBEV focus originally had only Apodemus mice, bank voles appeared later, reached 20.5% positivity and raised the positivity in small rodents from 4% to 10.2% in 3 years. The results highlight the role of M. glareolus and of adult rodents in maintaining the TBEV in nature.