Comparative efficacy of rHaa86 and rBm86 against Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus

Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus are the most economically important tick species in India and other tropical and subtropical regions of the world and transmit pathogens causing animal and human diseases. We demonstrated that vaccination of animal by rHaa86 could be used for the control of both H. a. anatolicum and R. (B.) microplus infestations. By comparing the efficacy of rHaa86 and rBm86, it was observed that vaccine based on rHaa86 will be more effective in controlling homologous challenge infestations (68·7% against larvae and 45·8% against adults). The results of this trial demonstrated that species-specific antigens are the better choice for vaccine development and could serve as an effective tool for the integrated control of H. a. anatolicum.     

Crimean-congo haemorrhagic fever: a seroepidemiological and tick survey in the Sultanate of Oman

Summary In 1995 and 1996, 4 persons from the Sultanate of Oman were confirmed with clinical Crimean-Congo haemorrhagic fever (CCHF). To assess the prevalence of CCHF virus infection in Oman, a convenience sample of imported and domestic animals from farms, abattoirs and livestock markets was examined by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to CCHF virus. Ticks were collected from selected animals, identified, pooled by species, host and location and tested for evidence of infection with CCHF virus by antigen-capture ELISA. Serum samples from individuals working in animal and nonanimal contact-related jobs were also tested for CCHF antibodies. Serological evidence of infection was noted in 108 (22%) of 489 animals. Most of the ticks collected (618 of 912) from all species of sampled livestock were Hyalomma anatolicum anatolicum , a competent vector and reservoir of CCHF virus. 243 tick pools were tested for CCHF antigen, and 19 pools were positive. Of the individuals working in animal contact-related jobs, 73 (30.3%) of 241 non-Omani citizens and only 1 (2.4%) of 41 Omani citizens were CCHF antibody-positive. Butchers were more likely to have CCHF antibody than persons in other job categories. The presence of clinical disease and the serological results for animals and humans and infected Hyalomma ticks provide ample evidence of the presence of CCHF virus in yet another country in the Arabian Peninsula.     

Isolation and characterization of salivary antigens from Hyalomma anatolicum anatolicum

This study demonstrates the involvement of a large number of salivary proteins in the acquisition of resistance to Hyalomma anatolicum anatolicum. Using immunoblotting, sera from hypersensitized rabbits were shown to react with nine proteins in the saliva and 17 in salivary gland extracts (SGE) from 96 h fed female ticks. The salivary antigens had molecular weights in the range of 14 400 to 130 000. All the antigens identified in the saliva and 12 of the SGE antigens were glycoprotein in nature and a majority of them appeared to be common to different stages of feeding. In addition antigen I (molecular weight 130 000) showed acid phosphatase and antigen III (molecular weight 96 000) showed both non-specific esterase and aminopeptidase activity. Three high molecular weight proteins isolated from saliva (antigen I, antigen II--molecular weight 103 000 and antigen III), gave immediate hypersensitivity reactions in intradermal inoculation into rabbits which had previously been exposed to ticks. Antigens II and III also elicited a strong delayed hypersensitivity reaction. These results may help to explain the nature of the immune mechanisms which effect resistance against H. a. anatolicum.     

Cloning, expression and immunoprotective efficacy of rHaa86, the homologue of the Bm86 tick vaccine antigen, from Hyalomma anatolicum anatolicum

The Bm86 homologue of Hyalomma anatolicum anatolicum Izatnagar isolate was cloned and expressed in methylotropic yeast Pichia pastoris as intracellular, glycosylated and particulated form. It was named as rHaa86, the first recombinant protein of H. a. anatolicum . Seven epidermal growth factor-like domains predicted in Haa86 were structurally similar with that of its Bm86 counterpart. The identity between the corresponding EGF like domains of Bm86 and Haa86 were ranging from 51·3% to 78·3%. The molecular weight of the rHaa86 was 120–140 kDa, with possible 50–70 kDa glycosylation. The purified rHaa86 was characterized immunologically and evaluated for its immunoprotective potential against homologous challenge infestation in three groups of cross-bred calves. The immediate rejection percentage of females of H. a. anatolicum was 36 5%, 12·4% and 10·1% fed on immunized (group 1), adjuvant control (group 2) and untreated control (group 3) calves, respectively. The percent rejection of female ticks fed on immunized calves was 24·1% and 26·4% higher than for the ticks fed on control groups 2 and 3, respectively ( P <  0·05). The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 58·0%, 9·0%, 5·0% and 61·6%, respectively. The mean reproductive index of females fed on group 1 calves was significantly lower ( P <  0·05) than the females fed on the control groups and 44% reduction in the number of engorged larvae was recorded from the group 1 calves. The data demonstrated that rHaa86 antigen based vaccine could serve as one of the effective components in the integrated control of H. a. anatolicum.     

Murine extramedullary erythropoiesis induced by tick infestation

Tick saliva contains molecules that modulate the haemostasis, pain/itch responses, wound healing and immune defences of the host. Using BALB/c mice that were each infested with 10 nymphs of Dermacentor andersoni Stiles (Acari: Ixodidae), an attempt has now been made to determine the influence of tick infestation on the expression of leucocyte adhesion molecules in the host. The ticks became fully engorged by the fourth to sixth day of infestation. On the fourth day of infestation, the results of flow cytometry indicated that 2% of the host's splenocytes were expressing high levels of CD49 (alpha4 integrin of VLA-4) and low levels of CD11a (alphaL subunit of the integrin LFA-1). By the eighth day of infestation, 30% of the hosts' splenocytes had this phenotype and were negative for the lineage markers CD3e (T-lymphocytes), DX5 (natural-killer cells of a BALB/c lineage), B220 (B-lymphocytes), CD11b (monocytes/macrophages, granulocytes, natural-killer cells, activated T-lymphocytes, and B-1 cells) and CD11c (myeloid and splenic dendritic cells). Histological examination of the spleens from infested mice revealed disruption of the white-pulp/red-pulp demarcations and the presence of a large number of basophilic normoblasts. The CD11a(lo) population of splenocytes from the tick-infested mice was positive for TER-119 but negative for CD3, B220, CD11b and Gr, confirming that the splenocytes were members of the erythroid lineage. These results indicate that, within 8 days of their initiation, the tick infestations induced extramedullary erythropoiesis in the spleens of their murine hosts.     

Crimean-Congo hemorrhagic fever virus in ticks collected from humans, livestock, and picnic sites in the hyperendemic region of Turkey

During June and July 2007, about 3125 adult ticks were collected from humans, animals, and vegetation in a hyperendemic region (Sivas and Tokat) of Turkey. A total of 2193 ticks were pooled in 225 pools and screened for the Crimean Congo hemorrhagic fever virus (CCHFV) presence by antigen-capture enzyme-linked immunosorbent assay. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). The dominant tick species was found to be Hyalomma marginatum with the following infestation rates in human, cattle and sheep, respectively: 47.43%, 66.07%, and 30.12%. Maximum likelihood estimation values of CCHFV in H. marginatum ticks collected from human, cattle, and sheep were 0.91% (CI 0.05-4.42), 2.10% (CI 1.12-3.64), and 3.11% (CI 1.18-6.87), respectively. CCHFV antigens were also demonstrated in Hyalomma excavatum, Haemaphysalis parva, and Boophilus annulatus ticks collected from cattle and Rhipicephalus bursa ticks from sheep. Our results suggest that the studied area might maintain its endemic properties in the near future unless effective tick control measures are implemented.     

Spotted fever rickettsiae in ticks from the northern Sinai Governate, Egypt.

A field study was initiated in 1988 to investigate whether spotted fever group rickettsiae occur in geographic areas in Egypt that are adjacent to an area in the southern Israeli Negev that has a defined focus of spotted fever disease. Ticks were collected from dogs, sheep, and camels at four study sites in the northern Sinai. Tick hemolymph was processed for rickettsial detection by staining with fluorescein isothiocyanate-conjugated antibody to Rickettsia rickettsii. Of the 442 hemolymphs examined, 15 contained immunofluorescent rickettsiae. Eight hemolymph test-positive (HT+) ticks were Rhipicephalus sanguineus removed from dogs; the other HT+ ticks comprised three Hyalomma species, H. anatolicum, H. impeltatum, and H. dromedarii. Both HT+ and HT- ticks were tested for rickettsial DNA using the polymerase chain reaction (PCR). Eight of 10 HT+ field-collected ticks were PCR positive (PCR+). All laboratory colony R. rickettsii-infected ticks were PCR+. No HT- ticks from field or laboratory isolates were PCR+.     

Molecular detection of spotted fever group rickettsiae associated with ixodid ticks in Egypt

Tick-borne diseases comprise a complex epidemiological and ecological network that connects the vectors, pathogens, and a group of host species. The aim of this study was to identify bacteria from the genus Rickettsia associated with ixodid ticks infesting camels and cows in Egypt. Ticks were collected from 6 different localities: Qina, Giza, Qalet El Nakhl, New Valley, El Arish, and Minufia, from July to October 2008. Species were identified using PCR, followed by sequencing. The gltA and rOmpA genes were used for the initial detection of Rickettsia spp. Further characterization of positive samples utilized primers targeting rOmpB, sca4, and intergenic spacers (mppA-purC, dksA-xerC, and rpmE-tRNA(fMet)). Cows were infested with Hyalomma anatolicum excavatum and Boophilus annulatus. Camels were infested with Hyalomma dromedarii, H. impeltatum, and H. marginatum marginatum. Approximately 57.1% of H. dromedarii ticks collected from Qalet El Nakhl were infected with Rickettsia africae, exhibiting 99.1-100% identity to reference strains. Within H. impeltatum, 26.7% and 73.3% of ticks from El Arish were infected with R. africae and R. aeschlimannii, with 98.3-100% and 97.9-100% identity, respectively. Furthermore, 33.3% of H. marginatum marginatum ticks in Qalet El Nakhl were infected with the same two species as H. impeltatum, demonstrating 99.1-100% and 99.3-100% identity, respectively. By comparing percent identities and phylogenetic relationships, R. africae is identified for the first time in Egypt, in addition to R. aeschlimannii, which exhibits 100% identity with the Stavropol strain in GenBank. In conclusion, the obtained data underscore the medical and veterinary importance of tick-borne rickettsioses, which necessitate further investigation by authorities in Egypt. Moreover, additional characterization of these rickettsial isolates should be performed to designate their strains, using a polyphasic strategy combining genotypic and phenotypic tests, to facilitate their deposition in the rickettsial collection of the WHO and/or ATCC.     

Dermacentor andersoni: effects of repeated infestations on lymphocyte proliferation, cytokine production, and adhesion-molecule expression by BALB/c mice.

The effects of repeated infestations with Dermacentor andersoni nymphs on the lymphocyte functions of BALB/c mice were investigated. The in-vitro proliferation responses to concanavalin-A or salivary-gland molecules, the production of cytokines, and the expression of two adhesion molecules-leucocyte function-associated antigen-1 (LFA-1) and very late activation-4 (VLA-4)-were all studied. In addition, the ability of salivary-gland extract or saliva from D. andersoni to modulate expression of lymphocyte adhesion molecules in vitro was determined. The proliferative responses of T-lymphocytes to concanavalin-A were significantly suppressed after first and second infestations, and significant increases in lymphocyte proliferation in the presence of tick salivary-gland antigen were observed in infested mice. After two infestations, production of interleukin-2 was significantly decreased but that of interferon-gamma remained unchanged. Production of interleukin-4 and interleukin-10 was significantly enhanced in infested mice after both the first and second infestations. Expression of LFA-1 and VLA-4 by lymphocytes from infested mice was suppressed. Furthermore, both a salivary-gland extract and the saliva of D. andersoni reduced the in-vitro expression of both of these adhesion molecules by lymphocytes from tick-naive mice.     

A survey of ticks on farm animals in Libya.

Thirteen species of ixodid ticks and two species of argasid ticks were collected during a three-year survey of 58 farms in Libya. These included Boophilus annulatus, B. microplus, B. decoloratus, seven species of Hyalomma, Rhipicephalus sanguineus, Rh. evertsi, Rh. bursa, Argas persicus and Ornithodoros foleyi. This is the first recording of B. microplus, B. decoloratus and Rh. bursa in Libya. Of 20,391 animals examined by random sampling, 2020 (9.6%) had ticks; particularly common were Hy. dromedarii on camels, Hy. impeltatum on sheep and Hy. excavatum on cattle. The tick found most frequently overall was Hy. dromedarii.     

The development of Theileria annulata in the salivary glands of the vector tick Hyalomma anatolicum anatolicum

The development at 28 degrees C of Theileria annulata (Hissar) in the salivary glands of its tick vector, Hyalomma anatolicum anatolicum, was studied using Giemsa-stained smears, methyl green-pyronin-stained preparations of whole glands, and electron microscopy. Nymphs which had engorged on T. annulata-infected calves showed kinetes in the haemolymph from Day 7 to Day 14 post engorgement, when all ticks had completed moult. Intracellular sporonts were observed within salivary gland acini from Day 7 onwards and these developed by rapid nuclear division and cytoplasmic proliferation to form primary sporoblasts. Further development was stimulated by feeding on a rabbit or by incubation at 36 degrees C. The primary sporoblasts appeared to become organized into membrane-bound subunits. Within 48 hours of attachment to the host, or 72 hours of 36 degrees C incubation, these units dissociated to form secondary sporoblasts. The final phase of development resulted in the progressive formation of discrete, uninuclear sporozoites within these secondary sporoblasts. No morphological differences were observed in parasite maturation between the fed and incubated groups although development was retarded by at least 24 hours in the latter. In the incubated group there was also a marked decrease in the degree of synchrony of development which resulted in fewer sporozoites being present at any one time.     

Experimental studies on the replication and transmission of Crimean-Congo hemorrhagic fever virus in some African tick species

Seven African tick species were studied as potential vectors of Crimean-Congo hemorrhagic fever (CCHF) virus. Engorged nymphae of 4 ixodid species, Hyalomma marginatum rufipes, H. truncatum, Rhipicephalus evertsi mimeticus, and Amblyomma hebraeum, were inoculated intracoelomically with CCHF virus and assayed for virus content at varying times post-inoculation. The virus replicated in all 4 species, reaching maximum titers of 4.6-5.5(10) fluorescence focus units per ml on days 5-9 post-inoculation. Virus titers declined up to the molt, but increased slightly on emergence of adult ticks. Thereafter, virus titers declined progressively, but infectivity could still be detected in adult ticks for up to 205 days post-inoculation. Groups of H. m. rufipes, H. truncatum, and R.e. mimeticus infected adults were fed on susceptible sheep and successfully transmitted CCHF infection. CCHF virus was not isolated from pools of the larval and nymphal progeny of the female ticks nor did the larvae transmit infection to guinea pigs by bite. CCHF virus failed to replicate in adults and nymphae of 3 argasid tick species, Argas walkerae, Ornithodorus porcinus porcinus, and O. savignyi, after intracoelomic inoculation and could be reisolated from the ticks no later than 1 day post-inoculation. The results suggest that all ixodid ticks are capable of transmitting CCHF virus but argasid ticks do not appear to be capable of serving as vectors.     

Ticks infesting wild and domestic animals and humans of Sri Lanka with new host records

An island-wide collection of tick species infesting humans, domesticated and wild animals and questing ticks in domestic and peridomestic environments was carried out during 2009–2011. A total of 30,461 ticks were collected from 30 different hosts and free living stages from the ground. The collection consisted of 22 tick species from 30 different hosts recording 12 tick species from humans, 19 from domesticated animals and 21 from wild animals, with a total of 97 new host records. The most common tick species on humans were Dermacentor auratus and Amblyomma testudinairum, while Haemaphysalis intermedia, Rhipicephalus microplus and Rhipicephalus sanguineus were common in domesticated and wild animals sharing 20 host species. Among the questing ticks, immature D. auratus was the most abundant. Humans and domesticated animals were mostly infested by the nymphal stages while adult ticks were found on wild animals. High number of new host records could be due to domestic animals picking tick species from wildlife and vise versa at the human/animal interface. Habitat destruction due to forest fragmentation has lead to wild animals roaming in urban and semi-urban neighbourhoods increasing the interactions of wild animals with domesticated animals. Wild animals play a significant role as a reservoir of many tick borne infections which can easily be spread to domesticated animals and then to humans via tick infestations. Data in this paper are useful for those interested in tick infesting wild and domestic animals and humans in describing the zoonotic potential of tick borne infections.     

Antigenic profile of Ixodes ricinus: effect of developmental stage, feeding time and the response of different host species

Antigens recognized by host species in response to ectoparasite infestation have been widely reported. Although differences in the immune responses of different host species have been described, only a very few of these studies compare the range of antigens recognized by different host species in response to infestation. We used Western blot analysis to investigate antigenic responses of different host species that were repeatedly infested with Ixodes ricinus ticks. Antigenic profiles of larval and nymphal whole tick homogenates were compared with the respective salivary gland extract (SGE) samples using sera from rabbits repeatedly infested with either adults, nymphs or larvae. SGE samples were also analysed using sera from hamsters infested with adults, nymphs or larvae. Sera from BALB/C mice, Apodemus flavicollis (yellow-necked mouse) or Clethrionomys glareolus (bank vole) repeatedly infested with larvae were used to compare the antigenic profiles of SGE and larval homogenate samples. We also investigated different sources of tick antigens, using rabbit sera, by comparing midgut extracts from female adult ticks and SGE from unfed ticks and from ticks throughout the 6-day feeding period with whole tick homogenates of female and male adults, nymphs and larvae. The pattern of antigenic tick-molecules recognized by infested host species varies with the period of feeding, developmental stage and the particular host species parasitized.     

广西地区家畜牛、羊体表硬蜱情况调查及种类鉴定

目的 掌握广西地区牛羊场硬蜱种类及其分子特征,了解该地区蜱类的分类地位,为养殖户防控硬蜱及蜱媒传染病提供参考依据。方法 本实验于2019年1月至2021年11月,采用动物体表法采集寄生的蜱类;提取蜱基因组,PCR扩增3种硬蜱的线粒体16S rDNA 和COI基因片段,并进行同源性分析。基于邻接法,用MEGA6.0软件分别构建系统发生树,进行遗传进化分析。结果 牛羊体表上共采集蜱2 030只,隶属1科2 属3种,其中微小扇头蜱1 968只,长角血蜱49只,具角血蜱13只。PCR扩增微小扇头蜱、长角血蜱和具角血蜱16S rDNA 和COI基因片段长度分别是460 bp和710 bp左右,分别与GenBank 中已登录的相应种类相似较高且在一个进化分支上。结论 广西地区牛羊场优势蜱种是微小扇头蜱且存在长角血蜱和具角血蜱。三蜱种16S rDNA 和COI序列存在多样性和地域差异性。     

Distribution of Crimean-Congo hemorrhagic fever viral antibody in Senegal: environmental and vectorial correlates

The spatial pattern in Senegal of Crimean-Congo hemorrhagic fever (CCHF) virus IgG antibody prevalence in human and sheep was determined as was the relative abundance of potential tick vectors. A systematic, country-wide serological survey of sheep demonstrated that 10.4% of sheep exhibited IgG to CCHF virus. Sexes were infected equally. Antibody prevalence increased with age from 2.1% during the first year to 18.2% among sheep greater than or equal to 3 years of age. IgG prevalence was highest in the northern, arid Sahelian zone, averaging 75.7% seropositivity, and decreased to zero in the southern, moister Sudano-Guinean and Guinean zones. Human IgG prevalence ranged from 21% to less than 1% among the 8 sites that were sampled throughout the country, being greatest in the arid north and least in the south. Hyalomma ssp. ticks predominated in those biotopes where antibody prevalence was highest. The abundance of Hyalomma ticks may be the proximal determinant of endemic transmission.     

Immunologic relations between cattle and ticks, specifically between cattle and Boophilus microplus]

In the present investigation, it has been demonstrated that cattle become resistant to ticks after several heavy infestations, particularly with B. microplus. During development of the infestations, antibodies against salivary glands of B. microplus were detected using 2 techniques: indirect immunofluorescence and immunoelectrophoresis. There is a positive causal relationship between antibody titer and resistance development. Two precipitating systems against B. microplus in infested cattle and 7 systems in immunized rabbits were studied. The systems 1 and 2 are similar in cattle and rabbits, but system 2 does not show any specificity, as it has been detected in cattle completely lacking tick infestations. Two one-day calves were treated with the antigen of B. microplus by injection of salivary glands and repeated infestations with a small number of larvae. They developed a pronounced resistance to the usual subsequent infestations by the ticks of the same species. Specific antibodies were found before the first usual infestation. This suggests that they might be responsible for resistance.     

Intrahepatic CD8+ T-lymphocyte response is important for therapy-induced viral clearance in chronic hepatitis B infection

BACKGROUND/AIMS: To determine which immune cells contribute to HBV-clearance during antiviral therapy, we performed a longitudinal analysis of intrahepatic immune cells during interferon-alpha therapy of chronic HBV-patients using the FNAB technique. METHODS: Twenty chronic HBeAg+-patients were treated with pegylated alpha-interferon combined with lamivudine or placebo for 52 weeks. FNAB and blood specimens were obtained at week 0, 2, 8 and 52. CD4+- and CD8+ T-lymphocytes, CD56+ cells, IFNgamma and granzyme B (GrB) were immunocytochemically quantified. RESULTS: The relative numbers of CD56+ cells and CD8+ T-lymphocytes were significantly higher in FNAB compared to blood at all time-points. Responders (n=9) exhibited significant increases in intrahepatic CD8+ and CD8+GrB+ lymphocytes, a small elevation in CD8+IFNgamma+ T-lymphocytes, no change in CD4+ T-lymphocytes, and a decrease in intrahepatic CD56+ cells during the first weeks of therapy. In non-responders (n=11) no significant changes in CD4+- and CD8+ T-lymphocytes and an increase in intrahepatic and CD56+ cells were observed during therapy. CONCLUSIONS: The intrahepatic CD8+ T-lymphocyte, but not the CD4+ T-lymphocyte or NK/NKT-cell response, is important for HBV clearance during interferon-alpha therapy, and the antiviral effect may be mediated by both cytolytic and non-cytolytic mechanisms.     

Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses

Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.