Ticks and tick-borne diseases from Mallorca Island, Spain.

Ixodid ticks are obligate blood-feeding arthropods and important vectors of pathogens. In Mallorca, almost no data on the tick fauna are available. Herein, we investigated ticks and tick-borne pathogens in ticks collected from dogs, a cat and humans in Mallorca as result of a citizen science project. A total of 91 ticks were received from German tourists and residents in Mallorca. Ticks were collected from March to October 2023 from dogs, cat and humans, morphologically and genetically identified and tested for pathogens by PCRs. Six tick species could be identified: Ixodes ricinus (n = 2), Ixodes ventalloi (n = 1), Hyalomma lusitanicum (n = 7), Hyalomma marginatum (n = 1), Rhipicephalus sanguineus s.l. (n = 71) and Rhipicephalus pusillus (n = 9). Rhipicephalus sanguineus s.l. adults were collected from dogs and four females from a cat and the 16S rDNA sequences identified it as Rh. sanguineus s.s. Hyalomma lusitanicum was collected from 1 human, 1 dog and 5 specimens were collected from the ground in the community of Santanyi, together with one H. marginatum male. This is the first report of Hyalomma marginatum in Mallorca. Both I. ricinus were collected from humans and I. ventalloi female was collected from a dog. All ticks tested negative for Anaplasma phagocytophilum, Coxiella spp., Francisella spp., and piroplasms. In 32/71 (45%) specimens of Rh. sanguineus s.s., Rickettsia spp. could be detected and in 18/32 (56.2%) sequenced tick DNAs R. massiliae was identified. Ixodes ventalloi female and both I. ricinus tested positive in the screening PCR, but the sequencing for the identification of the Rickettsia sp. failed.

Tick species from cattle in the Adama Region of Ethiopia and pathogens detected.

Ticks will diminish productivity among farm animals and transmit zoonotic diseases. We conducted a study to identify tick species infesting slaughter bulls from Adama City and to screen them for tick-borne pathogens. In 2016, 291 ticks were collected from 37 bulls in Adama, which were ready for slaughter. Ticks were identified morphologically. Total genomic DNA was extracted from ticks and used to test for Rickettsia spp. with real-time PCR. Species identification was done by phylogenetic analysis using sequencing that targeted the 23S-5S intergenic spacer region and ompA genes. Four tick species from two genera, Amblyomma and Rhipicephalus, were identified. Amblyomma cohaerens was the dominant species (n = 241, 82.8%), followed by Amblyomma variegatum (n = 22, 7.5%), Rhipicephalus pulchellus (n = 19, 6.5%), and Rhipicephalus decoloratus (n = 9, 3.0%). Among all ticks, 32 (11%) were positive for Rickettsia spp. and 15 (5.2%) of these were identified as R. africae comprising at least two genetic clades, occurring in A. variegatum (n = 10) and A. cohaerens (n = 5). The remainder of Rickettsia-positive samples could not be amplified due to low DNA yield. Furthermore, another 15 (5.2%) samples carried other pathogenic bacteria: Ehrlichia ruminantium (n = 9; 3.1%) in A. cohaerens, Ehrlichia sp. (n = 3; 1%) in Rh. pulchellus and A. cohaerens, Anaplasma sp. (n = 1; 0.5%) in A. cohaerens, and Neoehrlichia mikurensis (n = 2; 0.7%) in A. cohaerens. All ticks were negative for Bartonella spp., Babesia spp., Theileria spp., and Hepatozoon spp. We reported for the first time E. ruminatium, N. mikurensis, Ehrlichia sp., and Anaplasma sp. in A. cohaerens. Medically and veterinarily important pathogens were mostly detected from A. variegatum and A. cohaerens. These data are relevant for a One-health approach for monitoring and prevention of tick-borne disease transmission.

First detection of Rickettsia conorii ssp. caspia in Rhipicephalus sanguineus in Zambia.

Ticks are important vectors for Rickettsia spp. of the spotted fever group all around the world. Rickettsia conorii is the etiological agent of boutonneuse fever in the Mediterranean region and Africa. Tick identification was based on morphological features and further characterized using the 16S rRNA gene. The ticks were individually tested using pan-Rickettsia real-time-PCR for screening, and 23S-5S intergenic spacer region, 16S rDNA, gltA, sca4, ompB, and ompA genes were used to analyze the Rickettsia positive samples. Rickettsia conorii ssp. caspia was detected in tick collected in Zambia for the first time, thus demonstrating the possibility of the occurrence of human disease, namely Astrakhan fever, due to this Rickettsia ssp. in this region of Africa. The prevalence of R. conorii ssp. caspia was 0.06% (one positive tick out of 1465 tested ticks) and 0.07% (one positive tick out of 1254 tested Rh. sanguineus).

Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects.

BACKGROUND: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. METHODS: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR-/-) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. RESULTS: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. CONCLUSIONS: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.

Rickettsiosis in Denmark: A nation-wide survey.

Rickettsia helvetica has been reported at varying prevalences in Danish and other European Ixodes ricinus populations. Though apparently widespread and with reported cases of human infection, the significance of the bacteria as a threat to public health remains unclear. We present a nation-wide survey of rickettsia in ticks, roe deer and humans in Denmark. Ticks were collected by flagging and screened for presence of rickettsial DNA by polymerase chain reaction. Sera from roe deer, hunters, neuroborreliosis patients and blood donors were analyzed for presence of anti-R. helvetica and Rickettsia felis antibodies by immunofluorescence microscopy. The Rickettsia minimum infection rate in ticks was 4.9 % (367/973 pools positive, 7510 ticks in total), with 3.9 % in nymphs and 9.3 % in adults. Rickettsia helvetica accounted for 4.17 % and Rickettsia monacensis for 0.03 %, 0.6 % comprised non-differentiable rickettsial DNA. The prevalence of antibodies against R. helvetica was 2.8 % (9/319) in roe deer, while no hunters (n = 536) or blood donors (n = 181) were positive. The prevalence of anti-R. helvetica antibodies among Lyme neuroborreliosis patients was 6 % (3/47), where it co-occurred with Anaplasma phagocytophilum. Based on our study autochthonous rickettsiosis is of limited concern to the public health in Denmark, but our finding of R. monacensis for the first time in Denmark illustrates the dynamic nature of tick-borne pathogens, emphasizing that continuous surveillance is necessary.

Rickettsia spp. in ticks (Acari: Ixodidae) from wild birds: First detection of Candidatus Rickettsia vini in Hesse, Germany.

Tick-borne diseases are recognized as a growing public health concern and cause significant issues in humans and animals by serving as dispersal agents of ticks and their associated pathogens. Birds contribute to the spread of ticks and tick-borne diseases, with migratory birds playing a role in regional dispersal. Only a few studies have been carried out in Germany so far to investigate tick fauna on birds and the pathogens they harbour. In 2017 and 2018, we collected ticks from a total of 358 birds in the northern region of the Federal State of Hesse, Germany. Most ticks were Ixodes ricinus larvae and nymphs (95.4%). However, two ornithophilic species were also collected: Ixodes frontalis (larvae, nymphs and females) and Ixodes arboricola (larvae and nymphs). Ticks were tested individually or in pools, depending on the tick life stage and species for each bird host, in order to detect tick-borne encephalitis (TBE) virus and Rickettsia spp. All samples tested negative for TBE virus. Of 684 investigated tick pools, 162 tested positive for Rickettsia spp. by screening PCR. Of these, 117 carried Rickettsia helvetica detected by specific real-time PCR. The samples which were negative in the R. helvetica PCR were further investigated using five targets (gltA, 16S, ompA IV, ompB, 23S-5S intergenic spacer region) using multi locus sequence typing and phylogenetic analysis. The obtained sequences showed strong phylogenetic relationship to Candidatus Rickettsia vini, which is most closely related to R. japonica and R. heilongjiangensis, and occur in I. arboricola. Three more sequences were determined as R. helvetica in I. ricinus and I. frontalis. This is the first detection of Ca. R. vini in Germany and the first 23S-5S data published for Ca. R. vini, a useful target for Rickettsia species identification.

Tick-borne lymphadenopathy (TIBOLA) acquired in Southwestern Germany.

BACKGROUND: Tick-borne lymphadenopathy (TIBOLA) was first described in 1997 in a patient in France. The causative agent, Rickettsia slovaca, is transmitted by Dermacentor ticks. CASE PRESENTATION: In southwestern Germany we encountered a patient with a tick bite at the dorsal scalp that resulted in an eschar and nuchal lymphadenopathy. Additionally, fever, malaise as well as elevated inflammatory markers and transaminases occurred. The characteristic clinical picture along with positive antibody testing for rickettsiae of the tick-borne spotted fever group strongly suggest the diagnosis TIBOLA. CONCLUSION: Human rickettsioses are emerging infections. Clinicians should be aware of TIBOLA as a newly described rickettsial disease. As in our case, TIBOLA may be encountered in regions/countries where R. slovaca and Dermacentor ticks are prevalent but autochthonous acquisition was not described before.

Impact of flavivirus vaccine-induced immunity on primary Zika virus antibody response in humans.

BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

Rickettsia diversity in southern Africa: A small mammal perspective.

Worldwide, including Africa, rickettsioses are recognized as emerging or re-emerging infections. To date, little is known about the diversity of Rickettsia species that are naturally associated with small mammals in southern Africa. The aim of the study was to screen a diversity of small mammals for the presence of rickettsial DNA. Animals were trapped at 38 localities in South Africa and Namibia. In total, 1616 ear-tissue samples from 23 species representing 17 genera were tested using real-time (rt)PCR and multi-locus sequence typing (MLST). Of the 1616 samples 251 (15.5%) were positive in an initial rtPCR. In 16 of the 23 investigated animal species rickettsial DNA was detected with an average prevalence of 15.7%. We herein describe for the first time four Rickettsia (R.) species known to be pathogenic for humans in rodents from South Africa, R. conorii, R. massiliae, R. felis and R. helvetica. In addition, by MLST and subsequent phylogenetic analyses so far undescribed Rickettsia species, Candidatus Rickettsia africaustralis, Candidatus Rickettsia rhabdomydis, and Candidatus Rickettsia muridii were confirmed. Further four new genotypes, genotype Rickettsia hofmannii, genotype Rickettsia stutterheimensis, genotype Rickettsia hogsbackensis and genotype Rickettsia kaalplaasensis, respectively, are described. The data indicate a surprisingly high diversity of Rickettsia in small mammals in South Africa and might indicate their possible role as reservoirs for Rickettsia. Ecological questions concerning their natural hosts such as small mammals, but also the role of livestock or pet animals, require further investigation. Particularly, data on the relevance of these rickettsiae for diseases in humans are of further interest.

Ixodes inopinatus - Occurring also outside the Mediterranean region.

We report the presence of Ixodes inopinatus and its sympatric occurrence with Ixodes ricinus in southeastern Germany, western Austria, and Romania. The identification of I. inopinatus was based on morphological and molecular 16S rRNA and 12S rRNA gene features. We also report the finding of Rickettsia monacensis and Rickettsia helvetica in I. inopinatus collected from a fox and a sheep in Romania. Although the vector competence of I. inopinatus for these pathogens remains to be proven, there is evidence of transstadial persistence, an important prerequisite for acting as a vector.

Detection of Rickettsia monacensis and Rickettsia amblyommatis in ticks collected from dogs in Costa Rica and Nicaragua.

The neotropical climate of Central America provides ideal conditions for ticks, which may transmit several human pathogens, including spotted-fever group Rickettsia. Dogs may act as sentinels or reservoirs for human tick-borne diseases due to shared tick species. Here, ticks were collected from 680 client-owned dogs in Nicaragua and Costa Rica, and a total of 316 tick pools were investigated for Rickettsia infection by quantitative real-time PCR (qPCR) targeting the gltA gene. Subsequently, up to six further genomic targets (16S rDNA, gltA, sca4, ompA, ompB and the 23S-5S intergenic spacer) were investigated for Rickettsia species determination. The predominant tick species was Rhipicephalus sanguineus sensu lato (s.l.) (19.9% of dogs infested in Costa Rica, 48.0% in Nicaragua), followed by Ixodes boliviensis (3.1% in Costa Rica / none in Nicaragua) and Amblyomma ovale (4.8% in Costa Rica, 0.9% in Nicaragua). In total, 22 of 316 tick pools containing 60 of 1023 individual ticks were Rickettsia-positive as determined by qPCR, resulting in a minimum infection rate (MIR) of 2.2%. In detail, MIR in Rh. sanguineus s.l. was 0.7% (7/281 pools), in I. boliviensis 33.3% (12/13 pools) and in A. ovale 9.7% (3/22 pools). For 11 of 12 positive I. boliviensis pools and one of six positive Rh. sanguineus s.l. pools, the species could be determined as R. monacensis. R. amblyommatis was identified in one Rh. sanguineus s.l. pool from Costa Rica and one A. ovale pool from Nicaragua. Nine of 12 R. monacensis-positive tick pools were collected in San Rafael de Heredia, Costa Rica, indicating a high local occurrence in this area. This study supports recent evidence that R. monacensis is present on the American continent. Its high local occurrence among dog-associated I. boliviensis, which may also parasitize humans, in Costa Rica gives cause for concern, as R. monacensis is also pathogenic to humans.

High seroprevalence for indigenous spotted fever group rickettsiae in forestry workers from the federal state of Brandenburg, Eastern Germany.

In the last decade six Rickettsia species, including Rickettsia slovaca have been characterized in Germany. All of these species could be linked to distinct clinical syndromes in humans. However, due to lack of seroepidemiological data an estimation of the prevalence and the public health impact of rickettsial infections in Germany is difficult. The aim of the present study was to determine the seroprevalence of spotted fever group (SFG) rickettsiae in a population with an elevated exposure risk to ticks. For that purpose, 559 sera of forestry workers in the federal state of Brandenburg, Eastern Germany, were screened for SFG-rickettsiae reactive IgG antibodies. Positive sera were subsequently titrated by microimmunofluorescence assay against R. helvetica, R. raoultii, R. felis, "R. monacensis" and R. slovaca. The total average IgG seroprevalence rate against SFG rickettsiae of 27.5% was found to be represented by 9.7% R. helvetica, 5% R. raoultii, 2.7% R. felis, 0.5% "R. monacensis" and 0.5% R. slovaca. The remaining 9.1% positive test results were of non-differentiable origin. IgG seroprevalences ranged from 11% to 55% in the different forestry districts. Older and male participants had a significantly higher probability for seropositivity and higher anti-rickettsia antibody titer level. In addition, the number of recent as well as the recalled lifetime tick bites was significantly associated with seropositivity and higher titers against SFG rickettsiae. In conclusion, we found an unexpected high total seroprevalence against SFG rickettsiae in forestry workers and serological evidence confirming the occurrence of R. raoultii, R. felis, "R. monacensis" and R. helvetica in the federal State of Brandenburg.

First detection of Hyalomma rufipes in Germany.

Hyalomma rufipes, a two-host tick, is the most widespread Hyalomma species in Africa. In December 2015, an ixodid tick male with an unusual morphology was detected on a horse in a stable near Mainz in the Federal State Rhineland-Palatine. For identification purposes, the tick was preserved in alcohol and sent to our laboratory. The morphology of the tick showed specific characteristics of H. rufipes. The 16S rDNA sequence of H. rufipes from Germany was identical to the corresponding 16S rDNA sequence of H. rufipes from Tanzania, and they both were closely related to Hyalomma marginatum. The tick was tested with a real-time PCR for rickettsiae and Crimean-Congo hemorrhagic fever (CCHF) virus with negative results.

Extensive Recruitment of Plasma Blasts to the Cerebrospinal Fluid in Toscana Virus Encephalitis.

An unexpectedly extensive recruitment of B cells and plasma blasts to the cerebrospinal fluid (CSF) in a patient with Toscana virus (TOSV) encephalitis is described. Acute infection by TOSV was demonstrated by serological methods and by detection of TOSV-specific nucleic acid in the CSF by real-time polymerase chain reaction and sequencing.

Serological differentiation of antibodies against Rickettsia helvetica, R. raoultii, R. slovaca, R. monacensis and R. felis in dogs from Germany by a micro-immunofluorescent antibody test.

BACKGROUND: Spotted Fever Group (SFG) Rickettsiae can cause febrile diseases with or without rash in humans worldwide. In Germany only limited data are available about their medical significance. Serological screening tests for antibodies against rickettsiae usually only distinguish between SFG and Typhus Group (TG) Rickettsiae due to the strong cross reactivities within the groups. Seroprevalence rates in dogs, as possible sentinels for tick-borne diseases, could serve as an indicator for the distribution of different Rickettsia species. METHODS: In this study, a micro-immunofluorescence assay (micro-IFA) was established for detection and differentiation of antibodies against five Rickettsia species in dogs (R. helvetica, R. raoultii, R. slovaca, R. monacensis and R. felis). Dogs that never left Germany (n = 605) previously investigated with an SFG-ELISA were included in this study and screened at a 1:128 dilution. Endpoint titres of fifty randomly selected seropositive samples of each of the five investigated regions in Germany were determined in order to allow a differentiation of the causative Rickettsia species. Sensitivity and specificity of the micro-IFA were compared with ELISA results of the previous study. RESULTS: A total of 93.9% of the dogs were positive for antibodies of the SFG Rickettsiae at the screening titer of 1:128. Differentiation of SFG Rickettsiae with the micro-IFA was possible in 70.4%, but in 29.6% of the cases the detected antibodies were not differentiable. Considering a clear differentiation by a twofold titre difference between observed reactions, the seroprevalence rates were 66.0% for R. helvetica, 2.8% for R. raoultii, 1.6% for R. slovaca, but no serological reaction could be clearly attributed to R. monacensis or R. felis. No statistically significant regional differences were found for R. helvetica, R. slovaca and R. raoultii comparing the five regions of Germany. Comparison of micro-IFA with ELISA revealed a sensitivity of 82.0% and a specificity of 83.8% for the Rickettsia SFG ELISA. CONCLUSIONS: The micro-IFA is a useful serological tool to differentiate antibodies against different Rickettsia species in dogs. Seroprevalence rates in dogs correspond to the prevalence rates and distribution of Rickettsia-carrying tick species.

Complete genome sequence of a chikungunya virus imported from bali to Germany.

Chikungunya virus (CHIKV) strain DH130003 was isolated from a traveler with Chikungunya fever returning from Bali to Germany. Although strains of the east-central/south African lineage bearing the A226V mutation have predominated in most parts of Asia since 2005, CHIKV DH130003 belongs to the Asian lineage.

Viral hemorrhagic fever cases in the country of Georgia: Acute Febrile Illness Surveillance Study results.

Minimal information is available on the incidence of Crimean-Congo hemorrhagic fever (CCHF) virus and hantavirus infections in Georgia. From 2008 to 2011, 537 patients with fever ≥ 38°C for ≥ 48 hours without a diagnosis were enrolled into a sentinel surveillance study to investigate the incidence of nine pathogens, including CCHF virus and hantavirus. Of 14 patients with a hemorrhagic fever syndrome, 3 patients tested positive for CCHF virus immunoglobulin M (IgM) antibodies. Two of the patients enrolled in the study had acute renal failure. These 2 of 537 enrolled patients were the only patients in the study positive for hantavirus IgM antibodies. These results suggest that CCHF virus and hantavirus are contributing causes of acute febrile syndromes of infectious origin in Georgia. These findings support introduction of critical diagnostic approaches and confirm the need for additional surveillance in Georgia.