Serosurvey and molecular detection of Crimean–Congo hemorrhagic fever virus (CCHFV) in northern Turkey

In this study, the hard ticks, whole blood and serum samples collected from small ruminants (sheep and goat) in middle Black Sea region of Turkey where Crimean-Congo hemorrhagic fever (CCHF) human cases were observed in the past years were surveyed for the presence of RNA and specific IgG antibodies from CCFH virus (CCHFV). CCHFV RNA was found in 30 of 255 tick pools (11.76%) and nine of 105 (8.57%) leucocyte samples. No CCHFV genomic RNA was detected from animals in Yildizeli and Vezirkopru. However, CCHFV RNA was found from animals in Gerze and Resadiye. Seventy-eight of 105 goat and sheep blood serum samples tested were antibody-positive for CCHFV by enzyme-linked immunosorbent assay (ELISA) (goat: 42/63; sheep: 36/42). Viral RNA was detected from tick samples in all of four provinces. Positivity rates for the provinces varied and were as follows: Gerze 13.04%, Resadiye 35.41%, Vezirkopru 1.61% and Yildizeli 6.06%. CCHFV genomic RNA was detected in four of seven tick species tested. These results suggest that these hard ticks may act as a reservoir for CCHFV in northern Turkey.     

New aspects about Crimean-Congo hemorrhagic fever (CCHF) cases and associated fatality trends: A global systematic review and meta-analysis

Crimean-Congo hemorrhagic fever (CCHF) is the most popular tick-borne disease causing by Crimean-Congo hemorrhagic fever virus (CCHFV). There are several valuable reviews considering some fields of the CCHF aspects. While there is no a systematic review about means and trends of CCHF cases and fatality rate, means and trends of CCHF cases and fatality rates of human occupations involved in CCHF. Therefore, this meta-analysis review performed to highlight and provide a global detailed of the above CCHF aspects. Among 398 collected papers, 173 papers were become this meta-analysis review. The study results confirm that an apparent increasing CCHF cases occurred through the past decades. The trends of annually and periodically CCHF cases and fatality rates were also increased. The means of annually and periodically CCHF cases and fatality rates were 57 and 432 cases, and 10 cases and 32.2 % and 49 cases and 28.8 %, respectively. The means of annually and periodically CCHF fatality rates are about one-tenth of CCHF human cases. The mean of CCHF fatality rates in Africa (22.0 %) is lower than Asia (33.5 %) and Europe (33.8 %). Among occupations involved in CCHF, agricultural (28.9 %), health-care (19.2 %) and slaughterhouse (16.7 %) workers, and farmers (13.9 %) had the maximum CCHF fatality rates in order. Based on literature review of CCHFV S-segment aspects, several clades and genotypes are reported to distribute in Africa, Asia and Europe regions. There are very wide fields to investigate the epidemiology characteristics of CCHFV clades, genotypes and their distribution in the future.     

Knowledge,beliefs, and practices regarding tick bites in the Turkish population in a rural area of the Middle Anatolian Region

The aim of this study is to determine the knowledge, beliefs, and practices of a population living in a rural area in regards to tick bites and Crimean-Congo hemorrhagic fever (CCHF). The study was conducted in a rural area located in Central Anatolia in the region of Eskişehir. A total of 1,500 individuals aged 20 years and older chosen by a stratified random sample were enrolled. A questionnaire was administered in person. In 264 (17.4%) participants, there was a history of being bitten by a tick. This rate was higher in older persons, males, married persons, and farmers. The most commonly reported protective behavior was wearing long sleeves and long pants when wandering in rural areas (65.1% of participants). The least commonly reported behavior was using insect repellent on skin or clothes (3.3% of participants). Only 799 participants (54%) had heard about CCHF as a disease associated with ticks. Females, those with primary school education, housewives, and male farmers had a high frequency of having heard about CCHF. Tick bites and CCHF are important public health problems, yet the current knowledge of these problems is not sufficient in populations living in rural areas of the Middle Anatolian Region of Turkey.     

Fine mapping epitope on glycoprotein Gc from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis, caused by CCHF virus (CCHFV) and which there are no diagnostic or therapeutic strategies. The C-terminus of glycoprotein (Gc) encoded by the CCHFV M gene is responsible for CCHFV binding to cellular receptors and acts as a neutralizing-antibody target. In this study, a modified biosynthetic peptide technique (BSP) was used to identify fine epitopes of Gc from the CCHFV YL04057 strain using rabbit antiserum against CCHFV-Gc. Six B cell epitopes (BCEs) and one antigenic peptide (AP) were identified: E1 (⁸⁸VEDASES⁹⁴), E2 (¹¹⁷GDRQVEE¹²³), E3 (²⁴¹EIVTLH²⁴⁶), AP-4 (²⁸¹DFQVYHVGNLLRGDKV²⁹⁶), E5a (³⁷⁰GDTP QLDL³⁷⁷), E5b (³⁷³PQLDLKAR³⁸⁰), and E6 (⁴⁴³HVRSSD⁴⁴⁸). Western blotting analysis showed that each epitope interacted with the positive serum of sheep that had been naturally infected with CCHFV, and the results were consistent with that of Dot-ELISA. The multiple sequence alignment (MSA) revealed high conservation of the identified epitopes among ten CCHFV strains from different areas, except for epitopes AP-4 and E6. Furthermore, three-dimensional structural modeling showed that all identified epitopes were located on the surface of the Gc “head” domain. These mapped epitopes of the CCHFV Gc would provide a basis for further increase our understanding CCHFV glycoprotein function and the development of a CCHFV epitope-based diagnostics vaccine and detection antigen.     

Crimean–Congo haemorrhagic fever virus: Past,present and future insights for animal modelling and medical countermeasures

Crimean–Congo haemorrhagic fever (CCHF) is a widespread tick‐borne viral zoonosis with a case‐fatality rate ranging from 9% to 50% in humans. Although a licensed vaccine to prevent infection by the CCHF virus (CCHFV) exists, its ability to induce neutralizing antibodies is limited and its efficacy against CCHFV remains undetermined. In addition, controlling CCHF infections by eradication of the tick reservoir has been ineffective, both economically and logistically, and the treatment options for CCHF remain limited. In this review, we first critically discuss the existing animal models to evaluate therapeutics for CCHF. We then review the therapeutic options for CCHF that have been investigated in human cases, followed by investigational drugs that have been evaluated in pre‐clinical studies. We highlight the importance of understanding human prognostic factors in developing an animal model for CCHF that recapitulates hallmarks of human disease and its implication for selecting therapeutic candidates.     

Molecular identification of tick-borne bacteria in wild animals and their ticks in Central Anatolia,Turkey

Wild animals fulfill an important mission in the ecology of tick-borne diseases as both suitable hosts to tick vectors and reservoirs of the pathogens. However, current data regarding the role of wild animals in the ecology of tick-borne pathogens is insufficient and more investigations are required. In this study, we investigated tick-borne bacterial pathogens in wild boar, hare, and fox and their ticks in Turkey. A total of 102 tick pools comprised of 445 ticks and blood samples were analyzed for the presence of bacterial DNA by PCRs targeted rickettsial gltA and ompA genes, 5S-23S rDNA gene for Borrelia spp., and msp4 gene for Anaplasma spp. As a result of PCR and sequence analyses, three pathogenic spotted fever group (SFG) rickettsiae, two SFG rickettsiae with unknown pathogenicity and one pathogenic Borrelia burgdorferi sensu lato were detected in samples obtained from wild animals. Rickettsia slovaca was detected in ticks (13.7% of tick pools) collected from wild boars and blood of a wild boar. In addition, the presences of R. hoogstraalii (19.6% of tick pools), R. aeschlimannii (5.8% of tick pools), R. sibirica subsp. mongolitimonae (1.9% of tick pools) and Candidatus R. goldwasserii (0.9% of tick pools) were detected in ticks collected from wild animals. Furthermore, B. burgdorferi sensu stricto was detected in a tick pool collected from a wild boar. This is the first report on the presence of Candidatus R. goldwasserii in Turkey. Consequently, this study shows that pathogenic Rickettsia and Borrelia species are circulating in Turkish wildlife and these pathogens can pose a threat to human health. Also, it has been determined that the investigated wild animals play a role as maintenance host for vector ticks; therefore, these animals must also be considered in the ecology of the mentioned pathogens.     

Pathogenesis of African swine fever virus in Ornithodoros ticks

African swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tick Ornithodoros porcinus porcinus. The pathogenesis of ASFV in O. porcinus porcinus ticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. Thus O. porcinus porcinus ticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition to O. porcinus porcinus, a number of North American, Central American and Caribbean species of Ornithodoros have been shown to be potential vectors of ASFV.     

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.     

Recent progress in molecular biology of Crimean-Congo hemorrhagic fever

Crimean–Congo hemorrhagic fever (CCHF) is a severe hemorrhagic fever in humans with a case fatality rate of up to 50%. A causative agent of CCHF is CCHF virus, which is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The virus is transmitted to humans through infected tick bites, squashed ticks or from direct contact with viremic animals or humans. Outbreaks of CCHF have been documented in Africa, the Middle East, Eastern Europe and Western Asia where the vector and/or reservoir ticks of Hyalomma spp. are distributed. Recent advances in molecular and biochemical analyses of CCHF virus revealed that the virus encodes larger proteins compared to other genus of Bunyavirus and the processing of viral proteins are complicated. Recent studies also showed that the CCHF viruses are relatively divergent in its genome sequence and the viruses are grouped in seven different clades. In general, these phylogenetic analyses based on sequences of S-RNA and L-RNA segment of CCHF viruses indicate that the seven clades correlate with their geographical location. The phylogenetic topology based on M-RNA segment sequences of CCHF viruses is different from those based on S-RNA and L-RNA segments. These analyses indicate that M-RNA segment reassortment events occur more frequently than those in S- and L-RNA segments.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Ixodid tick species attaching to dogs in Hungary

A survey was carried out to investigate the occurrence of hard tick species (Acari: Ixodidae) infesting domestic dogs in Hungary. Forty veterinary clinics from a wide geographical area were asked to collect hard ticks from dogs and to complete a questionnaire. In total, 25 veterinary clinics submitted 900 ticks from 310 dogs. Intensity of infestation ranged from one to 78 per dog. The most preferred sites of tick attachment in decreasing order were head, neck and legs. The majority of ticks (91.7%) were adults, which were identified to species level, the others were nymphs. Six species were found: Dermacentor reticulatus (48.9%), Ixodes ricinus (43.2%), Ixodes canisuga (5.6%), Haemaphysalis concinna (2%) and there was one specimen of both Dermacentor marginatus and Ixodes hexagonus. Single species infestation with I. ricinus or D. reticulatus was found on 145 (46.8%) and 120 animals (38.7%), respectively. Mixed infestation caused by these two species was detected on 24 dogs (7.7%). I. canisuga and H. concinna were found on seven and five dogs, respectively. D. reticulatus and I. ricinus were collected almost throughout the year, except for a single month. The activity peaks were in spring and in autumn for both species. Based on clinical signs, canine babesiosis was diagnosed by the veterinarians in 66 (21.3%) tick infested dogs. These dogs were more frequently infested with D. reticulatus than the others. Our data contribute to the understanding of geographical and seasonal distribution of ixodid tick species infesting dogs in Hungary. The implication of these data, for the risk of canine tick borne diseases is discussed.     

Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis

A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine.     

Infection by spotted fever rickettsiae in people, dogs, horses and ticks in Londrina, Parana State, Brazil

Spotted fever is a disease caused by bacteria from the genus Rickettsia of the spotted fever group (SFG). Rickettsia rickettsii is likely the main agent of Brazilian spotted fever (BSF). With the objective of gathering information on the circulation of SFG rickettsiae in Londrina, Parana state, ticks from dogs and horses and also blood from dogs, horses and humans were collected in a neighbourhood of the city which presented potential for circulation of rickettsiae between hosts and vectors. Amblyomma cajennense, Dermacentor nitens, and Rhipicephalus sanguineus ticks were subjected to Polymerase Chain Reaction targeting a fragment of the Rickettsia gltA gene. This specific gene encodes the enzyme citrate synthase of Rickettsia spp., and results on all ticks were negative. Human and animal sera were tested by Indirect Immunofluorescence Assay in which R. rickettsii and R. parkeri were used as antigens. Sera from 4.7% human, 2.7% canine and 38.5% equine were positive for R. rickettsii. For R. parkeri, 0.9% human, 2.7% canine and 11.5% equine samples were positive. All samples reactive to R. parkeri also reacted to R. rickettsii. An epidemiological questionnaire was applied, but there were no statistically significant results. Comparison of our serological results with previous studies in Brazil, among BSF endemic and non-endemic areas, indicates that there is no established rickettsial infection in the study area, a statement corroborated with our molecular analysis. Nonetheless, as humans of the present study are highly exposed to tick infestations, health education within the population is needed to obtain efficient tick control.     

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.     

Serological evidence of exposure to tick fever organisms in young cattle on Queensland dairy farms

OBJECTIVES: To compare the features of farms on which the exposure of young cattle to tick fever organisms is sufficient to ensure that immunity is high and the risk of clinical disease is low (endemic stability) with those of farms on which exposure is insufficient to induce widespread immunity (hence without endemic stability); to examine the relationships between the management of ticks and tick fever, and endemic stability to Babesia bovis, B. bigemina and Anaplasma marginale. DESIGN: Cross-sectional study of 874 cattle between the ages of 6 and 15 months on 64 dairy farms, from three centres in south-eastern Queensland (Mutdapilly, Dayboro and Kenilworth) and one centre in far-north Queensland (Malanda). PROCEDURE: Blood samples collected from between 5 and 20 calves from each farm were submitted for serological assay to determine exposure to B. bovis, B. bigemina and A. marginale. A questionnaire about the farm characteristics and the management of ticks and tick fever was completed with each farmer. RESULTS: On 73% of farms, confirmed clinical cases of tick fever were recalled by the farmer, indicating that tick fever was a threat on most farms. The majority of herds in the study (54 of 64) did not have sufficient numbers of seropositive animals aged between 6 and 15 months to have a low risk of tick fever. Region had an effect on the likelihood of endemic stability for all tick fever organisms. Cattle near Malanda in Far-north Queensland were more likely to be seropositive to B. bovis and B. bigemina. The method, strategy and intensity of tick control were not related to the likelihood of endemic stability when the effect of region was considered. The decision to leave a few ticks on cattle in an effort to induce endemic stability did increase the likelihood of endemic stability to A. marginale. However, in practical terms, it was ineffective, because only 26% of these farms had endemic stability against all three organisms. CONCLUSIONS: Given the low proportion of farms that have endemic stability to the tick fever organisms and the high likelihood of clinical disease, vaccination is recommended to protect dairy cattle from tick fever throughout the tick infested area of Queensland. However, further work is required to determine the economic value of vaccination, taking into account the costs of vaccination, of outbreaks and the protective value of vaccination.     

Evidence of Powassan/deer tick virus in adult black‐legged ticks (Ixodes scapularis) recovered from hunter‐harvested white‐tailed deer (Odocoileus virginianus) in Pennsylvania: A public health perspective

Studies reporting tick infection rates for Powassan virus (POWV), an emerging zoonotic arthropod‐borne pathogen responsible for POWV disease in the Commonwealth of Pennsylvania, are limited. To determine the presence and ascertain a statewide prevalence of POWV, ticks were collected from 9,912 hunter‐harvested white‐tailed deer (Odocoileus virginianus) heads presented to six regional Pennsylvania Game Commission Chronic Wasting Disease sampling stations in early December of 2013, 2014 and 2015. Of the 2,973 ticks recovered, 1,990 (66.9%) were identified as adult Ixodes scapularis (black‐legged tick). The 1,990 I. scapularis ticks were PCR‐tested for the presence of POWV. The ticks had a statewide Powassan/deer tick virus infection rate of 0.05%, providing evidence of this pathogen in Pennsylvania's adult I. scapularis ticks and supporting the need for more comprehensive pathogen prevalence assessment strategies, as well as increased public health awareness for this emerging zoonotic arthropod‐borne pathogen of public health concern.     

Experimental transmission of African swine fever virus by Ornithodoros savignyi (Audouin)

The 'sand tampan', Ornithodoros savignyi, is susceptible to oral infection with African swine fever (ASF) virus in the laboratory. Infected ticks can transmit the virus transstadially and are able to maintain it for at least 106 days. Transmission of ASF virus by infected ticks to healthy pigs was achieved on five separate occasions between 50 and 106 days after infection. Pigs infected in this way developed typical acute African swine fever. The distribution of O savignyi in Africa suggests that this tick could be a natural field vector of ASF.     

Species of Ticks on Camels and Their Seasonal Population Dynamics in Eastern Ethiopia

A study was conducted to identify the species of ticks found on camels (Camelus dromedarius) and their seasonal population dynamics in Eastern Ethiopia. Collection and identification of the ticks were undertaken at 2-month intervals from December 1997 to August 1999. On each occasion, all the visible adult ticks were collected from one side of the body of each of the same 17 camels. The most abundant species of ticks on the camels were Rhipiephalus pulchellus (85.2%), Hyalomma dromedarii (5.9%), Amblyomma gemma (4.0%) and Amblyomma variegatum (1.8%). The average tick load per camel was higher during rainy months than during dry months. The smallest number of ticks per camel was observed during the driest month (December), whereas the highest was recorded in the wettest month (August). Any strategy intended to mitigate problems of tick infestation of camels in this area should take into account the identified tick species and their season of abundance.     

Some observations on the sero-prevalence of heartwater and tick infestation in Zambian goats

A survey was carried out to define the distribution of heartwater in goats that originated from six districts in communal grazing semi-arid areas of Zambia. A total of 181 samples (40.1%) out of 451 serum samples from adult goats were positive for Ehrlichia ruminantium antibodies after screening using indirect MAP-1B antigen ELISA technique with statistically significant differences (P < 0.01) between the six districts. Out of 1 036 adult goats examined for tick infestation, 105 were infested by ticks, with Amblyomma species being the most dominant tick encountered. Amblyomma variegatum, which is the vector for heartwater transmission in Zambia constituted 42.4% of the tick species, identified. The overall tick infestation rate was 10% while the tick:goat ratio was 2.1:1. Amblyomma variegatum appears to be widespread throughout the study area, as are antibodies to E. ruminantium.