Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

Excretion Dynamics of Arboviruses in Mosquitoes and the Potential Use in Vector Competence Studies and Arbovirus Surveillance.

The increasing threat of arboviruses such as West Nile virus (WNV) and Usutu virus (USUV) requires the fast and efficient surveillance of these viruses. The examination of mosquitoes takes up an important part; however, these investigations are usually very time-consuming. An alternative sample type for arbovirus surveillance might be mosquito excreta. In order to determine the excretion dynamics under laboratory conditions, laboratory colonies of Aedes vexans and Culex pipiens biotype molestus were infected with WNV, USUV or tick-borne encephalitis virus (TBEV). After infection, the excreta were sampled and investigated for viral RNA. Excretion of viral RNA together with infectious blood meal could be detected up to five days after infection. Further excretion seemed to correlate with a disseminated infection in mosquitoes, at least after USUV infection. In addition, it could be determined that the amount of viral RNA in the excretions correlated positively with the viral load in the mosquito bodies. Overall, this study shows that the usage of mosquito excreta as a sample type for surveillance enables the detection of endemic viruses (WNV, USUV) as well as non-mosquito-borne viruses (TBEV). In addition, examination of viral shedding during vector competence studies can provide insights into the course of infection without sacrificing animals.

Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

First Detection of Hepatitis E Virus (Rocahepevirus ratti Genotype C1) in Synanthropic Norway Rats (Rattus norvegicus) in Romania.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with different viral genera and species reported in a wide range of animals. Rodents, particularly rats, carry the specific genus rat HEV (Rocahepevirus genus, genotype C1) and are exposed occasionally to HEV-3 (Paslahepevirus genus, genotype 3), a zoonotic genotype identified in humans and widely distributed in domestic and feral pigs. In this study, the presence of HEV was investigated in synanthropic Norway rats from Eastern Romania, in areas where the presence of HEV-3 was previously reported in pigs, wild boars and humans. Using methods capable of detecting different HEV species, the presence of HEV RNA was investigated in 69 liver samples collected from 52 rats and other animal species. Nine rat liver samples were identified as being positive for rat HEV RNA (17.3%). High sequence identity (85-89% nt) was found with other European Rocahepevirus. All samples tested from other animal species, within the same environment, were negative for HEV. This is the first study to demonstrate the presence of HEV in rats from Romania. Since rat HEV has been reported to cause zoonotic infections in humans, this finding supports the need to extend the diagnosis of Rocahepevirus in humans with suspicion of hepatitis.

A Search for Tick-Associated, Bronnoya-like Virus Spillover into Sheep.

Tick-borne diseases are responsible for many vector-borne diseases within Europe. Recently, novel viruses belonging to a new viral family of the order Bunyavirales were discovered in numerous tick species. In this study, we used metatranscriptomics to detect the virome, including novel viruses, associated with Ixodes ricinus collected from Romania and France. A bunyavirus-like virus related to the Bronnoya virus was identified for the first time in these regions. It presents a high level of amino-acid conservation with Bronnoya-related viruses identified in I. ricinus ticks from Norway and Croatia and with the Ixodes scapularis bunyavirus isolated from a tick cell line in Japan in 2014. Phylogenetic analyses revealed that the Bronnoya viruses' sub-clade is distinct from several Bunyavirales families, suggesting that it could constitute a novel family within the order. To determine if Bronnoya viruses could constitute novel tick-borne arboviruses, a Luciferase immunoprecipitation assay for detecting antibodies in the viral glycoprotein of the Romanian Bronnoya virus was used to screen sera from small ruminants exposed to tick bites. No positive serum was detected, suggesting that this virus is probably not able to infect small ruminants. This study represents the first serological investigation of mammalian infections with a Bronnoya-like virus and an initial step in the identification of potential new emergences of tick-borne arboviruses.

First data on bacteria associated with bat ectoparasites collected in Kharkiv oblast, Northeastern Ukraine.

BACKGROUND: Bats (Mammalia: Chiroptera) serve as natural reservoirs for many zoonotic pathogens worldwide, including vector-borne pathogens. However, bat-associated parasitic arthropods and their microbiota are thus far not thoroughly described in many regions across the globe, nor is their role in the spillover of pathogens to other vertebrate species well understood. Basic epidemiological research is needed to disentangle the complex ecological interactions among bats, their specific ectoparasites and microorganisms they harbor. Some countries, such as Ukraine, are particularly data-deficient in this respect as the ectoparasitic fauna is poorly documented there and has never been screened for the presence of medically important microorganisms. Therefore, the aims of this study were to provide first data on this topic. METHODS: A total of 239 arthropod specimens were collected from bats. They belonged to several major groups of external parasites, including soft ticks, fleas, and nycteribiid flies from six chiropteran species in Northeastern Ukraine. The ectoparasites were individually screened for the presence of DNA of Rickettsia spp., Anaplasma/Ehrlichia spp., Bartonella spp., Borrelia spp., and Babesia spp. with conventional PCRs. Positive samples were amplified at several loci, sequenced for species identification, and subjected to phylogenetic analysis. RESULTS: Rickettsia DNA was detected exclusively in specimens of the soft tick, Carios vespertilionis (7 out of 43 or 16.3%). Sequencing and phylogenetic analysis revealed high similarity to sequences from Rickettsia parkeri and several other Rickettsia species. Bacteria from the family Anaplasmataceae were detected in all groups of the ectoparasites (51%, 122/239 samples), belonging to the genera Anaplasma, Ehrlichia, and Wolbachia. The detection of Bartonella spp. was successful only in fleas (Nycteridopsylla eusarca) and bat flies (Nycteribia koleantii, N. pedicularia), representing 12.1% (29/239) of the collected ectoparasites. No DNA of Babesia or Borrelia species was identified in the samples. CONCLUSIONS: We report for the first time in Ukraine the molecular detection of several bacterial agents in bat ectoparasites collected from six species of bats. The data presented extend the knowledge on the distribution of ectoparasite species in bats and their involvement in potentially circulating agents pathogenic for humans and vertebrate animals.

West Nile virus in the Republic of Serbia-Diagnostic performance of five serological tests in dog and horse sera.

West Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of 'in-house' and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to 'gold standard' virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n = 184) and horses (n = 232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by 'in-house' ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by 'in-house' IFA to 32.5% by commercially available IFA and from 26.3% by 'in-house' IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between 'in-house' and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2  = 8.647, p = .003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. 'In-house' tests remain a valuable tool in early diagnostics of WNV.

Sympatric occurrence of Ixodes ricinus with Dermacentor reticulatus and Haemaphysalis concinna and the associated tick-borne pathogens near the German Baltic coast.

BACKGROUND: Ixodid ticks from the Northern Hemisphere have registered a northward expansion in recent years, and Dermacentor reticulatus is such an example in Europe, its expansion being considered a result of climate change alongside other factors. The aim of this study was to identify the composition of questing tick species and the associated pathogens at different sites near the German Baltic coast. METHODS: Questing ticks were collected monthly at four sites (May-November, 2020), mainly grasslands, and in October and November 2020 at a fifth site. Molecular screening of ticks for pathogens included RT-qPCR for the tick-borne encephalitis virus (TBEV), qPCR for Anaplasma phagocytophilum, PCR for Babesia species and Rickettsia species, and nested PCR for Borrelia species. RESULTS: Altogether 1174 questing ticks were collected: 760 Ixodes ricinus, 326 D. reticulatus and 88 Haemaphysalis concinna. The highest activity peak of I. ricinus and D. reticulatus was in May, in June for H. concinna while a second peak was observed only for I. ricinus and D. reticulatus in September and October, respectively. All samples tested negative for TBEV. For A. phagocytophilum, 1.5% of I. ricinus adults tested positive while the minimum infection rate (MIR) in nymphs was 1.3%. This pathogen was found in 0.6% of D. reticulatus. Babesia spp. were detected in I. ricinus (18.2% adults, 2.1% MIR in nymphs) and H. concinna (13.3% adults, 9.7% MIR in nymphs). Borrelia spp. were present only in I. ricinus (49.1% adults, 11.9% MIR in nymphs), while Rickettsia spp. were detected in I. ricinus (14% adults, 8.9% MIR in nymphs) and D. reticulatus (82%). Co-detection of pathogens was observed in 26.6% and 54.8% of positive I. ricinus adults and nymph pools, respectively, while one D. reticulatus tested positive for A. phagocytophilum and Rickettsia spp. The most common co-infection in I. ricinus adults was Babesia microti and Borrelia afzelii (12.3% of positive ticks). CONCLUSIONS: The results of this study confirm the northern expansion of D. reticulatus and H. concinna in Germany. The detailed data of the infection levels at each location could be useful in assessing the risk of pathogen acquisition following a tick bite.

Zoonotic pathogen screening of striped field mice (Apodemus agrarius) from Austria.

The striped field mouse (Apodemus agrarius) is known to carry several zoonotic pathogens, including Leptospira spp. and Dobrava-Belgrade orthohantavirus (DOBV). Since its first detection in 1996 in south-east Austria, the striped field mouse has further expanded its range in Austria. Here, we screened 35 striped field mice collected in an Austrian region near the Hungarian border for DOBV, Leptospira spp. and seven vector-borne pathogens. Hantavirus RT-PCR screening and DOBV IgG ELISA analysis led to the detection of two DOBV-positive striped field mice. The complete coding sequences of all three genome segments of both strains were determined by a combination of target enrichment and next-generation sequencing. Both complete coding S segment sequences clustered within the DOBV genotype Kurkino clade with the highest similarity to a sequence from Hungary. In one of 35 striped field mice, Leptospira borgpetersenii sequence type (ST) 146 was detected. Bartonella spp., Borrelia miyamotoi and Neoehrlichia mikurensis DNA was detected in four, one and two of 32 mice, respectively. Babesia, Anaplasma, Ehrlichia and Rickettsia specific DNA was not detected. Future investigations will have to determine the prevalence and invasion of these pathogens with the ongoing range expansion of the striped field mouse in Austria.

Anaplasma phagocytophilum and Anaplasma ovis-Emerging Pathogens in the German Sheep Population.

Knowledge on the occurrence of pathogenic tick-borne bacteria Anaplasma phagocytophilum and Anaplasma ovis is scarce in sheep from Germany. In 2020, owners from five flocks reported ill thrift lambs and ewes with tick infestation. Out of 67 affected sheep, 55 animals were clinically examined and hematological values, blood chemistry and fecal examinations were performed to investigate the underlying disease causes. Serological tests (cELISA, IFAT) and qPCR were applied to all affected sheep to rule out A. phagocytophilum and A. ovis as a differential diagnosis. Ticks were collected from selected pastures and tested by qPCR. Most animals (n = 43) suffered from selenium deficiency and endoparasites were detected in each flock. Anaplasma spp. antibodies were determined in 59% of examined sheep. Seventeen animals tested positive for A. phagocytophilum by qPCR from all flocks and A. phagocytophilum was also detected in eight pools of Ixodes ricinus. Anaplasma phagocytophilum isolates from sheep and ticks were genotyped using three genes (16S rRNA, msp4 and groEL). Anaplasma ovis DNA was identified in six animals from one flock. Clinical, hematological and biochemical changes were not significantly associated with Anaplasma spp. infection. The 16S rRNA analysis revealed known variants of A. phagocytophilum, whereas the msp4 and groEL showed new genotypes. Further investigations are necessary to evaluate the dissemination and health impact of both pathogens in the German sheep population particularly in case of comorbidities.

Borrelia burgdorferi Sensu Lato in Questing and Engorged Ticks from Different Habitat Types in Southern Germany.

Borrelia burgdorferi sensu lato (s.l.) causes the most common tick-borne infection in Europe, with Germany being amongst the countries with the highest incidences in humans. This study aimed at (1) comparing infection rates of B. burgdorferi s.l. in questing Ixodes ricinus ticks from different habitat types in Southern Germany, (2) analysing genospecies distribution by habitat type, and (3) testing tissue and ticks from hosts for B. burgdorferi s.l. Questing ticks from urban, pasture, and natural habitats together with feeding ticks from cattle (pasture) and ticks and tissue samples from wild boars and roe deer (natural site) were tested by PCR and RFLP for species differentiation. B. burgdorferi s.l. was found in 29.8% questing adults and 15% nymphs. Prevalence was lower at the urban sites with occurrence of roe deer than where these were absent. Borrelia burgdorferi s.l. DNA was found in 4.8% ticks from roe deer, 6.3% from wild boar, and 7.8% from cattle. Six genospecies were identified in unfed ticks: Borrelia afzelii (48.6%), Borrelia burgdorferi sensu stricto (16%), Borrelia garinii (13.2%), Borrelia valaisiana (7.5%), Borrelia spielmanii (6.2%), and Borrelia bavariensis (0.9%). This study shows high infection levels and a great diversity of Borrelia in questing ticks. The presence of roe deer seems to reduce B. burgdorferi s.l. infection rates in tick populations.

Global Distribution of Babesia Species in Questing Ticks: A Systematic Review and Meta-Analysis Based on Published Literature.

Babesiosis caused by the Babesia species is a parasitic tick-borne disease. It threatens many mammalian species and is transmitted through infected ixodid ticks. To date, the global occurrence and distribution are poorly understood in questing ticks. Therefore, we performed a meta-analysis to estimate the distribution of the pathogen. A deep search for four electronic databases of the published literature investigating the prevalence of Babesia spp. in questing ticks was undertaken and obtained data analyzed. Our results indicate that in 104 eligible studies dating from 1985 to 2020, altogether 137,364 ticks were screened with 3069 positives with an estimated global pooled prevalence estimates (PPE) of 2.10%. In total, 19 different Babesia species of both human and veterinary importance were detected in 23 tick species, with Babesia microti and Ixodesricinus being the most widely reported Babesia and tick species, respectively. Regardless of species, adult ticks with 2.60% had the highest infection rates, while larvae had the least with 0.60%. Similarly, female ticks with 4.90% were infected compared to males with 3.80%. Nested-polymerase chain reaction (PCR) 2.80% had the highest prevalence among the molecular techniques employed. In conclusion, results obtained indicate that Babesia species are present in diverse questing tick species at a low prevalence, of which some are competent vectors.

Tick species identification and molecular detection of tick-borne pathogens in blood and ticks collected from cattle in Egypt.

To address the lack of information on ticks infesting cattle in Egypt and the pathogens that they transmit, the current study aimed to (i) provide insight into tick species found on cattle in Egypt, (ii) identify the pathogens in ticks and their cattle hosts and (iii) detect pathogen associations in ticks and cattle. Tick samples and blood from their bovine hosts were collected from three different areas in Egypt (EL-Faiyum Oasis, Assiut Governorate and EL-Kharga Oasis). Tick species were identified by morphology and by sequence analysis of the cytochrome C oxidase subunit 1 (cox1) gene. Tick pools and blood samples from cattle were screened by the Reverse Line Blot hybridization (RLB) assay for the simultaneous detection of tick-borne pathogens, including Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia spp., as well as the tick endosymbiont Midichloria mitochondrii. The RLB results were confirmed with specific conventional and semi-nested PCRs followed by sequencing. In total, 570 ticks (males, females and nymphs) were collected from 41 heads of cattle. Altogether 398 ticks belonged to the genus Hyalomma (397 Hyalomma excavatum and one Hyalomma scupense) while 172 ticks were identified as Rhipicephalus annulatus. Pooled H. excavatum ticks tested positive for several protozoa and bacteria with different minimum infection rates (MIRs): Theileria annulata (18.1 %), Babesia occultans (1.8 %), Anaplasma marginale (28.5 %), Anaplasma platys (0.25 %), Midichloria mitochondrii (11.6 %), Ehrlichia chaffeensis-like (1.8 %) and Ehrlichia minasensis (1 %). In R. annulatus, several agents were identified at different MIRs: T. annulata (2.3 %), B. bovis (0.6 %), A. marginale (18.0 %), A. platys (1.2 %), M. mitochondrii (2.9 %), E. minasensis (0.6 %). Pathogens co-detection in tick pools revealed A. marginale and T. annulata in 13.3 % samples followed by the co-detection of A. marginale and M. mitochondrii (8.4 %). In addition, triple co-detection with A. marginale, T. annulata and M. mitochondrii were found in 5.3 % of the tick pools. In cattle, the most common coinfection was with A. marginale and T. annulata (82.9 %) followed by the coinfection between A. marginale, T. annulata and B. bovis (4.9 %), A. marginale and B. bigemina (2.4 %) and finally the coinfection between T. annulata and B. occultans (2.4 %). Anaplasma platys, Babesia occultans, and E. minasensis were detected for the first time in Egypt in both cattle and ticks. These findings should be taken in consideration regarding human and animal wellbeing by the public health and veterinary authorities in Egypt.

Transstadial Transmission and Replication Kinetics of West Nile Virus Lineage 1 in Laboratory Reared Ixodes ricinus Ticks.

West Nile virus (WNV) is a mosquito-borne agent that has also been isolated from several tick species. Vector competence of Ixodes ricinus, one of the most common tick species in Europe, has been poorly investigated for WNV to date. As such, to evaluate the vector competence, laboratory reared Ixodes ricinus nymphs were in vitro fed with WNV lineage 1 infectious blood, allowed to molt, and the resulting females artificially fed to study the virus transmission. Furthermore, we studied the kinetics of WNV replication in ticks after infecting nymphs using an automatic injector. Active replication of WNV was detected in injected nymphs from day 7 post-infection until 28 dpi. In the nymphs infected by artificial feeding, the transstadial transmission of WNV was confirmed molecularly in 46.7% of males, while virus transmission during in vitro feeding of I. ricinus females originating from infected nymphs was not registered. The long persistence of WNV in I. ricinus ticks did not correlate with the transmission of the virus and it is unlikely that I. ricinus represents a competent vector. However, there is a potential reservoir role that this tick species can play, with hosts potentially acquiring the viral agent after ingesting the infected ticks.

Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt.

BACKGROUND: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. RESULTS: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.

Pathogenicity of West Nile Virus Lineage 1 to German Poultry.

West Nile virus (WNV) is a mosquito-borne virus that originates from Africa and at present causes neurological disease in birds, horses, and humans all around the globe. As West Nile fever is an important zoonosis, the role of free-ranging domestic poultry as a source of infection for humans should be evaluated. This study examined the pathogenicity of an Italian WNV lineage 1 strain for domestic poultry (chickens, ducks, and geese) held in Germany. All three species were subcutaneously injected with WNV, and the most susceptible species was also inoculated via mosquito bite. All species developed various degrees of viremia, viral shedding (oropharyngeal and cloacal), virus accumulation, and pathomorphological lesions. Geese were most susceptible, displaying the highest viremia levels. The tested waterfowl, geese, and especially ducks proved to be ideal sentinel species for WNV due to their high antibody levels and relatively low blood viral loads. None of the three poultry species can function as a reservoir/amplifying host for WNV, as their viremia levels most likely do not suffice to infect feeding mosquitoes. Due to the recent appearance of WNV in Germany, future pathogenicity studies should also include local virus strains.

Prevalence and molecular characterization of ticks and tick-borne pathogens of one-humped camels (Camelus dromedarius) in Nigeria.

BACKGROUND: Ticks are hematophagous arthropods responsible for maintenance and transmission of several pathogens of veterinary and medical importance. Current knowledge on species diversity and pathogens transmitted by ticks infesting camels in Nigeria is limited. Therefore, the aim of this study was to unravel the status of ticks and tick-borne pathogens of camels in Nigeria. METHODS: Blood samples (n = 176) and adult ticks (n = 593) were collected from one-humped camels (Camelus dromedarius) of both sexes in three locations (Kano, Jigawa and Sokoto states) in north-western Nigeria and screened for the presence of Rickettsia spp., Babesia spp., Anaplasma marginale, Anaplasma spp. and Coxiella-like organisms using molecular techniques. All ticks were identified to species level using a combination of morphological and molecular methods. RESULTS: Ticks comprised the three genera Hyalomma, Amblyomma and Rhipicephalus. Hyalomma dromedarii was the most frequently detected tick species (n = 465; 78.4%) while Amblyomma variegatum (n = 1; 0.2%) and Rhipicephalus evertsi evertsi (n = 1; 0.2%) were less frequent. Other tick species included H. truncatum (n = 87; 14.7%), H. rufipes (n = 19; 3.2%), H. impeltatum (n = 18; 3.0%) and H. impressum (n = 2; 0.3%). The minimum infection rates of tick-borne pathogens in 231 tick pools included Rickettsia aeschlimannii (n = 51; 8.6%); Babesia species, (n = 4; 0.7%) comprising of B. occultans (n = 2), B. caballi (n = 1) and Babesia sp. (n = 1); Coxiella burnetii (n = 17; 2.9%); and endosymbionts in ticks (n = 62; 10.5%). We detected DNA of "Candidatus Anaplasma camelli" in 40.3% of the blood samples of camels. Other tick-borne pathogens including Anaplasma marginale were not detected. Analysis of risk factors associated with both tick infestation and infection with Anaplasma spp. in the blood indicated that age and body condition scores of the camels were significant (P < 0.05) risk factors while gender was not. CONCLUSIONS: This study reports low to moderate prevalence rates of selected tick-borne pathogens associated with camels and their ticks in north-western Nigeria. The presence of zoonotic R. aeschlimannii emphasizes the need for a concerted tick control programme in Nigeria.

West Nile Virus Lineage 2 Vector Competence of Indigenous Culex and Aedes Mosquitoes from Germany at Temperate Climate Conditions.

West Nile virus (WNV) is a widespread zoonotic arbovirus and a threat to public health in Germany since its first emergence in 2018. It has become of particular relevance in Germany in 2019 due to its rapid geographical spread and the detection of the first human clinical cases. The susceptibility of indigenous Culex pipiens (biotypes pipiens and molestus) for a German WNV lineage 2 strain was experimentally compared to that of Serbian Cx. pipiens biotype molestus and invasive German Aedes albopictus. All tested populations proved to be competent laboratory vectors of WNV. Culex pipiens biotype pipiens displayed the highest transmission efficiencies (40.0%-52.9%) at 25 °C. This biotype was also able to transmit WNV at 18 °C (transmission efficiencies of 4.4%-8.3%), proving that temperate climates in Central and Northern Europe may support WNV circulation. Furthermore, due to their feeding behaviors, Cx. pipiens biotype molestus and Ae. albopictus can act as "bridge vectors", leading to human WNV infections.

Comparison of vector competence of Aedes vexans Green River and Culex pipiens biotype pipiens for West Nile virus lineages 1 and 2.

West Nile virus (WNV), a zoonotic arbovirus, has recently established an autochthonous transmission cycle in Germany. In dead-end hosts like humans and horses the WNV infection may cause severe symptoms in the central nervous system. In nature, WNV is maintained in an enzootic transmission cycle between birds and ornithophilic mosquitoes. Bridge vector species, such as members of the Culex pipiens complex and Aedes spp., also widely distributed in Germany, might transmit WNV to other vertebrate host species. This study determined and compared the vector competence of field-collected northern-German Cx. pipiens biotype pipiens and laboratory-reared Ae. vexans Green River (GR) for WNV lineage 1 (strain: Magpie/Italy/203204) and WNV lineage 2 (strain: "Austria") under temperatures typical for northern Germany in spring/summer and autumn. For assessment of vector competence, 7- to 14-day-old female mosquitoes were offered a WNV containing blood meal via Hemotek membrane feeding system or cotton-stick feeding. After incubation at 18°C respectively 24°C for 14 days engorged female mosquitoes were salivated and dissected for determination of infection, dissemination and transmission rates by reverse transcriptase quantitative real-time PCR (RT-qPCR). Both Ae. vexans GR and Cx. pipiens biotype pipiens were infected with both tested WNV strains and tested 14 days post-inoculation. Disseminated infections were detected only in Ae. vexans GR incubated at 18°C and in Cx. pipiens pipiens incubated at 24°C after infection with WNV lineage 1. Transmission of WNV lineage 1 was detected in Cx. pipiens pipiens incubated at 24°C. These results indicate that Cx. pipiens pipiens from Northern Germany may be involved in the transmission of WNV, also to dead-end hosts like humans and horses.

Borrelia miyamotoi and Borrelia burgdorferi (sensu lato) identification and survey of tick-borne encephalitis virus in ticks from north-eastern Germany.

BACKGROUND: Ixodes ricinus is the most common tick species in Europe and the main vector for Borrelia burgdorferi (sensu lato) and tick-borne encephalitis virus (TBEV). It is involved also in the transmission of Borrelia miyamotoi, a relapsing fever spirochete that causes health disorders in humans. Little is known regarding the circulation of Borrelia species and the natural foci of TBEV in north-eastern Germany. The goal of this study was to investigate the infection rates of Borrelia spp. and of TBEV in I. ricinus ticks from north-eastern Germany. METHODS: Ticks were collected by flagging from 14 forest sites in Mecklenburg-Western Pomerania between April and October 2018. RNA and DNA extraction was performed from individual adult ticks and from pools of 2-10 nymphs. Real time reverse transcription PCR (RT-qPCR) targeted the 3' non-coding region of TBEV, while DNA of Borrelia spp. was tested by nested PCR for the amplification of 16S-23S intergenic spacer. Multilocus sequence typing (MLST) was performed on B. miyamotoi isolates. RESULTS: In total, 2407 ticks were collected (239 females, 232 males and 1936 nymphs). Female and male I. ricinus ticks had identical infection rates (both 12.1%) for Borrelia spp., while nymphal pools showed a minimum infection rate (MIR) of 3.3%. Sequencing revealed four Borrelia species: B. afzelii, B. garinii, B. valaisiana and B. miyamotoi. Borrelia afzelii had the highest prevalence in adult ticks (5.5%) and nymphs (MIR of 1.8%). Borrelia miyamotoi was identified in 3.0% of adults and registered the MIR of 0.8% in nymphs. Borrelia valaisiana was confirmed in 2.5% adult ticks and nymphs had the MIR of 0.7%, while B. garinii was present in 1.1% of adults and showed a MIR of 0.1% in nymphs. The MLST of B. miyamotoi isolates showed that they belong to sequence type 635. No tick sample was positive after RT-qPCR for TBEV RNA. CONCLUSIONS: The prevalence of B. miyamotoi in I. ricinus ticks registered similar levels to other reports from Europe suggesting that this agent might be well established in the local tick population. The detection of B. burgdorferi (s.l.) indicates a constant circulation in tick populations from this region.