Zdenek Hubálek

Novel Flavivirus or New Lineage of West Nile Virus, Central Europe

Rabensburg virus, isolated from Culex pipiens mosquitoes in central Europe, represents a new lineage of West Nile virus or a novel flavivirus of the Japanese encephalitis virus group.

Isolation of Brucella microti from Soil

To the Editor: Brucella microti is a recently described Brucella species (1) that was isolated in 2000 from systemically infected common voles (Microtus arvalis) in South Moravia, Czech Republic. The organism is characterized by rapid growth on standard media and high metabolic activity, which is atypical for Brucella (2). The biochemical profile of B. microti is more similar to that of Ochrobactrum spp., of which most species are typical soil bacteria. n nOn the basis of the close phylogenetic relationship of Brucella spp. and Ochrobactrum spp. and the high metabolic activity of B. microti, we hypothesized that this Brucella species might also have a reservoir in soil. To test this hypothesis, we investigated 15 soil samples collected on December 11, 2007, from sites in the area where B. microti was isolated from common voles in 2000 (2). Ten of the samples were collected from the surface and at a depth of up to 5 cm near different mouse burrows 5 m apart. The remaining 5 samples were collected from an unaffected area without clinical cases of vole infection. The pH of soil samples ranged from 5.9 to 6.3. No frosts were recorded before the time of collection. n nTo specifically detect B. microti in soil samples, we have developed a PCR that targets a genomic island of 11 kb (H.C. Scholz et al., unpub. data) that is unique for B. microti. Briefly, primers Bmispec_f (5′-AGATACTGGAACATAGCCCG-3′) and Bmispec_r (5′-ATACTCAGGCAGGATACCGC-3′) were used to amplify a 510-bp fragment of the genomic island. PCR conditions were denaturation at 94°C for 5 min, followed by 29 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Total DNA was prepared from 0.5 g of each soil sample by using the MO BIO Ultra Clean Soil DNA Kit (Dianova, Hamburg, Germany). DNA was eluted with 50 μL of double-deionized water of which 2 μL was used in PCRs. Template DNA of B. microti CCM 4915T was used as a positive control. Type strains of all recognized Brucella species, 1 strain of each biovar of all species, and type strains of 11 Ochrobactrum species were used as negative controls. n nIn this PCR, 5 of 15 soil samples and the positive control were positive for the 510-bp fragment; other Brucella spp. and Ochrobactrum spp. were negative. Of the 5 positive samples, 3 were collected from surface soil collected near mouse burrows. However, the remaining 2 positive samples were collected from the unaffected and supposedly negative-control area. n nFor direct cultivation of Brucella spp. from soil, 2 g each of 2 selected PCR-positive samples with the highest amplification rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. Of a serial dilution in PBS (100–10–4), 100 μL was plated onto Brucella agar (Merck, Darmstadt, Germany) supplemented with 5% (vol/vol) sheep blood (Oxoid, Wesel, Germany) and Brucella selective supplement (Oxoid) and incubated at 37°C. Twenty suspicious colonies from the 100 dilution plate of 1 soil sample were subcultivated on Brucella selective agar. Two of the subcultivated bacteria (BMS 17 and BMS 20) reacted positively with monospecific anti-Brucella (M) serum. Both isolates were positive in the B. microti–specific PCR. Sequencing of the 510-bp fragments from both strains (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:AM943814,term_id:189086082}}AM943814 and {type:entrez-nucleotide,attrs:{text:AM943815,term_id:189086083}}AM943815) and comparison with the known nucleotide sequence of B. microti showed 100% identity. n nTo confirm that strains BMS 17 and BMS 20 were B. microti, these strains were subjected to multilocus sequence analysis and multilocus variable number of tandem repeat analysis (MLVA) as described (1,3–5). Multilocus sequence typing profiles of these strains were identical to the type strain B. microti CCM 4915T and strain CCM 4916. MLVA showed that these strains also clustered with B. microti strains CCM 4915T and CCM 4916, with identical panel 1 and panel 2A genotypes but a different panel 2B genotype. n nIn summary, we successfully isolated B. microti from soil samples collected at the same site 7 years after primary isolation of this novel species from common voles. B. microti could still be isolated from the same soil samples 6 months after storage at 4°C. This finding indicates long-term survival of B. microti in soil; thus, soil might function as a reservoir of infection. Identification of B. microti as a potential soil bacterium is consistent with Brucella spp. whole genome sequencing data, in particular with the genome sequence of B. suis, which exhibits fundamental similarities with plant pathogens such as Agrobacterium spp. and Rhizobium spp. (6). Whether soil is the primary habitat of B. microti or other vectors, such as nematodes, remains to be investigated.

Arboviruses pathogenic for domestic and wild animals.

The objective of this chapter is to provide an updated and concise systematic review on taxonomy, history, arthropod vectors, vertebrate hosts, animal disease, and geographic distribution of all arboviruses known to date to cause disease in homeotherm (endotherm) vertebrates, except those affecting exclusively man. Fifty arboviruses pathogenic for animals have been documented worldwide, belonging to seven families: Togaviridae (mosquito-borne Eastern, Western, and Venezuelan equine encephalilitis viruses; Sindbis, Middelburg, Getah, and Semliki Forest viruses), Flaviviridae (mosquito-borne yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile, Usutu, Israel turkey meningoencephalitis, Tembusu and Wesselsbron viruses; tick-borne encephalitis, louping ill, Omsk hemorrhagic fever, Kyasanur Forest disease, and Tyuleniy viruses), Bunyaviridae (tick-borne Nairobi sheep disease, Soldado, and Bhanja viruses; mosquito-borne Rift Valley fever, La Crosse, Snowshoe hare, and Cache Valley viruses; biting midges-borne Main Drain, Akabane, Aino, Shuni, and Schmallenberg viruses), Reoviridae (biting midges-borne African horse sickness, Kasba, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki, equine encephalosis, Peruvian horse sickness, and Yunnan viruses), Rhabdoviridae (sandfly/mosquito-borne bovine ephemeral fever, vesicular stomatitis-Indiana, vesicular stomatitis-New Jersey, vesicular stomatitis-Alagoas, and Coccal viruses), Orthomyxoviridae (tick-borne Thogoto virus), and Asfarviridae (tick-borne African swine fever virus). They are transmitted to animals by five groups of hematophagous arthropods of the subphyllum Chelicerata (order Acarina, families Ixodidae and Argasidae-ticks) or members of the class Insecta: mosquitoes (family Culicidae); biting midges (family Ceratopogonidae); sandflies (subfamily Phlebotominae); and cimicid bugs (family Cimicidae). Arboviral diseases in endotherm animals may therefore be classified as: tick-borne (louping ill and tick-borne encephalitis, Omsk hemorrhagic fever, Kyasanur Forest disease, Tyuleniy fever, Nairobi sheep disease, Soldado fever, Bhanja fever, Thogoto fever, African swine fever), mosquito-borne (Eastern, Western, and Venezuelan equine encephalomyelitides, Highlands J disease, Getah disease, Semliki Forest disease, yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile encephalitis, Usutu disease, Israel turkey meningoencephalitis, Tembusu disease/duck egg-drop syndrome, Wesselsbron disease, La Crosse encephalitis, Snowshoe hare encephalitis, Cache Valley disease, Main Drain disease, Rift Valley fever, Peruvian horse sickness, Yunnan disease), sandfly-borne (vesicular stomatitis-Indiana, New Jersey, and Alagoas, Cocal disease), midge-borne (Akabane disease, Aino disease, Schmallenberg disease, Shuni disease, African horse sickness, Kasba disease, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki disease, equine encephalosis, bovine ephemeral fever, Kotonkan disease), and cimicid-borne (Buggy Creek disease). Animals infected with these arboviruses regularly develop a febrile disease accompanied by various nonspecific symptoms; however, additional severe syndromes may occur: neurological diseases (meningitis, encephalitis, encephalomyelitis); hemorrhagic symptoms; abortions and congenital disorders; or vesicular stomatitis. Certain arboviral diseases cause significant economic losses in domestic animals-for example, Eastern, Western and Venezuelan equine encephalitides, West Nile encephalitis, Nairobi sheep disease, Rift Valley fever, Akabane fever, Schmallenberg disease (emerged recently in Europe), African horse sickness, bluetongue, vesicular stomatitis, and African swine fever; all of these (except for Akabane and Schmallenberg diseases) are notifiable to the World Organisation for Animal Health (OIE, 2012).

Absence of Lyme Disease Spirochetes in Larval Ixodes ricinus Ticks

To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.

Birds and Influenza H5N1 Virus Movement to and within North America

TOC Summary: Migratory birds are unlikely introductory hosts for this highly pathogenic virus in its present form into North America.

Serologic survey of potential vertebrate hosts for West Nile virus in Poland.

A survey for antibodies to West Nile virus (WNV; genus ,Flavivirus) was carried out by plaque-re-duction neutralization microtesting in 78 horses, 20 domestic chickens, and 97 wild birds belonging to 10 species from different areas in Poland. Specific antibodies were detected in five juvenile (hatching-year) birds collected in 2006: three white storks (Ciconia ciconia) in a wildlife rehabilitation center (5.4% of all examined storks; the antibody titers in each bird were 1:320, 1:160, and 1:20), one free-living mute swan (Cygnus olor; the titer was 1:20), and one hooded crow (Corvus corone cornix; the titer 1:20) in a wildlife rehabilitation center; thus the overall seropositivity to WNV was 5.2% among all the birds sampled. These data do not rule out the presence of WNV activity in Poland with 100% certainty, but they indicate a significant trace that demands verification. In addition, one black-headed gull (Larus ridibundus) had neutralizing antibodies for the Usutu Flavivirus, the first case recorded in Poland.

Putative New West Nile Virus Lineage in Uranotaenia unguiculata Mosquitoes, Austria, 2013

West Nile virus (WNV) is becoming more widespread and markedly effecting public health. We sequenced the complete polyprotein gene of a divergent WNV strain newly detected in a pool of Uranotaenia unguiculata mosquitoes in Austria. Phylogenetic analyses suggest that the new strain constitutes a ninth WNV lineage or a sublineage of WNV lineage 4.

Tick-borne Encephalitis Virus in Horses, Austria, 2011

An unexpectedly high infection rate (26.1%) of tick-borne encephalitis virus (TBEV) was identified in a herd of 257 horses of the same breed distributed among 3 federal states in Austria. Young age (p<0.001) and male sex (p = 0.001) were positively associated with infection.

Vector-borne disease intelligence: strategies to deal with disease burden and threats

Owing to the complex nature of vector-borne diseases (VBDs), whereby monitoring of human case patients does not suffice, public health authorities experience challenges in surveillance and control of VBDs. Knowledge on the presence and distribution of vectors and the pathogens that they transmit is vital to the risk assessment process to permit effective early warning, surveillance, and control of VBDs. Upon accepting this reality, public health authorities face an ever-increasing range of possible surveillance targets and an associated prioritization process. Here, we propose a comprehensive approach that integrates three surveillance strategies: population-based surveillance, disease-based surveillance, and context-based surveillance for EU member states to tailor the best surveillance strategy for control of VBDs in their geographic region. By classifying the surveillance structure into five different contexts, we hope to provide guidance in optimizing surveillance efforts. Contextual surveillance strategies for VBDs entail combining organization and data collection approaches that result in disease intelligence rather than a preset static structure.

Rickettsiae in questing Ixodes ricinus ticks in the Czech Republic.

Tick-borne rickettsiae are an important topic in the field of emerging infectious diseases. In the study, we screened a total of 1473 field-collected Ixodes ricinus ticks (1294 nymphs, 99 males, and 80 females) for the presence of human pathogenic rickettsiae (Rickettsia helvetica, R. monacensis, Candidatus Neoehrlichia mikurensis, and Anaplasma phagocytophilum) in natural and urban ecosystems using molecular techniques. The minimum infection rate (MIR) for Rickettsia spp. was found to be 2.9% in an urban park and 3.4% in a natural forest ecosystem; for Candidatus Neoehrlichia mikurensis, we observed MIRs of 0.4% in the city park and 4.4% in the natural habitat, while for A. phagocytophilum the MIR was 9.4% and 1.9%, respectively. Our study provides the first data on the occurrence of human pathogenic rickettsiae in questing I. ricinus ticks in the Czech Republic.