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.     

Pathogenicity of endophytic entomopathogenic fungi to Ornithodoros erraticus and Ornithodoros moubata (Acari: Argasidae)

The argasid ticks O. erraticus and O. moubata are of great medical and veterinary importance because they are vectors of the African swine fever virus and several species of human relapsing fever borreliae. Biocontrol of these ticks using entomopathogenic fungi has not been previously reported. We examined the pathogenicity to different developmental stages of these two argasids of six strains of the fungal species Beauveria bassiana (strains Bb1764 and Bb2157), Lecanicillium lecanii (strains Ll586, Ll618 and Ll3047) and Tolypocladium cylindrosporum (strain Tc3398). Three strains, Bb1764, Bb2157, and Tc3398, caused in Spanish O. erraticus mean mortality rates between 34.4% and 62% in 14-28 days post-inoculation. Additionally, Bb2157 also induced in African O. moubata mean mortality rates of 31.9%. The remaining strains caused lower mortality rates and were not considered effective. This is the first study in which some strains of entomopathogenic fungi are found to be effective against argasid ticks of the genus Ornithodoros, and its results might justify further efforts towards the application of entomopathogenic fungal strains as anti-argasid biocontrol agents.     

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.     

Cloning and characterization of a plasminogen-binding enolase from the saliva of the argasid tick Ornithodoros moubata

Significant amounts of enolase have recently been found in the saliva of the argasid tick Ornithodoros moubata, raising the question as to what the function of enolase in the tick–host interface is. Enolase is a multifunctional glycolytic enzyme known to act as a plasminogen receptor on cellular surfaces, promoting fibrinolysis and extracellular matrix degradation. Fibrinolysis could be important for ticks to dissolve clots that might be formed during feeding as well as to prevent clotting of the ingested blood meal in the tick midgut. Additionally, enolase-mediated extracellular matrix degradation could contribute to the tick feeding lesion. Moreover, previous observations suggested an additional antihaemostatic role for O. moubata enolase as a P-selectin antagonist ligand. Accordingly, the aim of the present study was to investigate the potential role of the O. moubata salivary enolase as a plasminogen receptor and P-selectin ligand, and to evaluate its potential as an antigen target for anti-O. moubata vaccines. The study included the cloning, sequencing and recombinant production of the O. moubata enolase, plasminogen binding and activation assays, P-selectin binding assays, animal immunization trials, and RNAi knockdown of the enolase gene. Here we confirmed that enolase is secreted to the saliva of the tick and provide convincing evidence for a role of this salivary enolase as a plasminogen receptor, most likely stimulating host fibrinolysis and maintaining blood fluidity during tick feeding. The RNAi experiments and immunization trials indicated that enolase could be also involved in the regulation of tick reproduction, suggesting new potential control strategies. Finally, the P-selectin binding experiments demonstrated that this enolase is not a P-selectin ligand.     

Antigens from the midgut membranes of Ornithodoros erraticus induce lethal anti-tick immune responses in pigs and mice

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In southern Europe O. erraticus lives in close association with swine on free-range pig farms. Application of acaricides for the eradication of O. erraticus from pig farms is inefficient. This is the reason why we tried to develop an anti-O. erraticus vaccine as alternative method of control. Accordingly, we were prompted to investigate the protective possibilities of a midgut membrane extract from the parasite (GME) that has not been studied hitherto. Administration of the GME with Freund's adjuvants (FAs) to pigs and mice induced a protective response able to kill 80% of the immature forms of the parasite in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. The action of the vaccine is the result of damage to the midgut wall of the argasid, and, in mice, it has been shown that this damage is mediated by activation of the complement system. In pigs, the administration of GME with alum, instead of with FAs, reduced the degree of protection. The protective antigens of the GME were expressed by all the developmental stages examined and are probably proteins from the luminal membrane of midgut epithelial cells. These antigens were seen to be more abundant in recently fed parasites than in fasting specimens, suggesting that their expression is induced after blood ingestion.     

Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.     

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.     

Distribution and biology of Ornithodoros erraticus in parts of Spain affected by African swine fever

Ornithodoros erraticus was found in 30.7 per cent, 35.0 per cent and 71.0 per cent of the pig-pens sampled in the provinces of Salamanca, Badajoz and Huelva in which African swine fever is a problem in the rearing of Iberian pigs. Between 38 and 65 per cent of the pig-pens in these areas are now abandoned and their populations of O erraticus are extinct or becoming so because they can no longer feed on pigs, which in Spain are their main hosts. The abandonment of pig-pens has resulted in the elimination of most soft ticks infected with the virus of African swine fever, and means that the distribution of ticks is now irregular and focal. Another factor affecting their distribution is the kind of soil on which the pig-pens are located. In abandoned pig-pens, the adults and large nymphs survive for about five years or longer when animals occasionally enter them. Hungry tick populations may transmit African swine fever when feeding in winter, whereas the populations that have continuous access to pigs do not feed until the pig-pens reach a temperature of 13 to 15 degrees C. In the latter populations, each stage exhibits a single annual peak of activity, which implies that the development from larva to adult takes two to three years. Pigs may die as a result of the bites, but on no occasion were 100 per cent of the fasting ticks seen to feed, even though they had the opportunity of doing so. This may hinder the eradication of this soft tick from infested pig-pens.     

Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus (Argasidae)

Ornithodoros erraticus is known to transmit the virus that causes the highly contagious disease, African Swine Fever, in Spain. As part of the disease eradication campaign, an ELISA test to detect specific antibodies against the tick was developed. The ELISA, using salivary gland preparations as an antigen, showed high sensitivity and was able to detect as few as 10 adult ticks. The specific antibodies were detected in the sera 6 weeks after the primary infestation and strongly increased after the challenge. The utility of this test under field conditions was also tested.     

Ornithodoros coriaceus (pajaroello tick) as a vector of bluetongue virus

Preliminary studies demonstrated that the argasid tick, Ornithodoros coriaceus Koch, could become infected with bluetongue virus (BTV). Ticks became infected after feeding through artificial membranes on BTV-infected suspensions of cell cultures, chicken embryos, and sheep blood. Ticks also became infected after natural feeding on viremic sheep (BTV serotype 17) and cattle (BTV serotype 11). Virus was recovered from the hemolymph and salivary glands of ticks which had ingested BTV either through an artificial membrane or by natural feeding on a host animal. Ticks infected with BTV serotype 13 were capable of transmitting the virus to a susceptible cow at 42 days after ingestion of virus-infected cultures, thus demonstrating the potential of the tick to serve as a biological vector of BTV.     

Molecular monitoring of African swine fever virus using surveys targeted at adult ornithodoros ticks: a re-evaluation of Mkuze game reserve, South Africa

The Mkuze Game Reserve (MGR), in north-eastern KwaZulu-Natal Province, South Africa is an African swine fever virus (ASF) controlled area. In a survey conducted in 1978, ASF prevalence in warthogs and Ornithodoros ticks in MGR was determined to be 2% and 0.06%, respectively. These values, acknowledged as being unusually low compared to other East and southern African ASF-positive sylvatic-cycle host populations, have not been assessed since. The availability of a sensitive PCR-based virus detection method, developed specifically for the sylvatic tampan host, prompted a re-evaluation of ASF virus (ASFV) prevalence in MGR ticks. Of the 98 warthog burrows inspected for Ornithodoros presence, 59 (60.2%) were found to contain tampans and tick sampling was significantly male-biased. Whilst gender sampling-bias is not unusual, the 27% increase in infestation rate of warthog burrows since the 1978 survey is noteworthy as it anticipates a concomitant increase in ASFV prevalence, particularly in light of the high proportion (75%) of adult ticks sampled. However, despite DNA integrity being confirmed by internal control amplification of the host 16S gene, PCR screening failed to detect ASFV. These results suggest that ASFV has either disappeared from MGR or if present, is localized, occurring at exceptionally low levels. Further extensive surveys are required to establish the ASFV status of sylvatic hosts in this controlled area.     

Parasites of domestic and wild animals in South Africa. XLVII. Ticks of tortoises and other reptiles

A total of 586 reptiles, belonging to 35 species and five subspecies, were examined in surveys aimed at determining the species spectrum and geographic distribution of ticks that infest them. Of these reptiles 509 were tortoises, 28 monitor or other lizards, and 49 snakes. Nine ixodid tick species, of which seven belonged to the genus Amblyomma, and one argasid tick, Ornithodoros compactus were recovered. Seven of the ten tick species are parasites of reptiles. Amongst these seven species Amblyomma marmoreum was most prevalent and numerous on leopard tortoises, Geochelone pardalis; Amblyomma nuttalliwas present only on Bell's hinged tortoises, Kinixys belliana; and most Amblyomma sylvaticum were collected from angulate tortoises, Chersina angulata. Amblyomma exornatum (formerly Aponomma exornatum) was only recovered from monitor lizards, Varanus spp.; most Amblyomma latum (formerly Aponomma latum) were from snakes; and a single nymph of Amblyomma transversale (formerly Aponomma transversale) was collected from a southern African python, Python natalensis. All 30 Namaqualand speckled padloper tortoises, Homopus signatus signatus, examined were infested with O. compactus. The seasonal occurrence of A. sylvaticum and the geographic distribution of this tick and of A. marmoreum, A. nuttalli, A. exornatum, A. latum and O. compactus are illustrated.     

Purification, N-terminal sequencing and diagnostic value of the major antigens of Ornithodoros erraticus and O. moubata

To enhance the specificity and sensitivity of serological detection of swine exposed to Ornithodoros erraticus or O. moubata, we purified the 158, 186, 215 and 260 kDa antigens from the former species and the designated (owing to their MW and charge) 19C, 17A, 20A1 and 20A2 antigens of the latter by HPLC and gel electroelution methods. All the O. erraticus antigens share epitopes and are difficult to purify individually by reverse phase and ion-exchange chromatography due to their molecular similarity. Tested individually by ELISA, all of them give the same optical densities (OD) with anti-O. erraticus sera, and these ODs are always lower with anti-immature than with anti-adult sera. Although immature and adult specimens have the same antigens, immature forms induce more anti-carbohydrate antibodies than adults. This is the reason for the lower ODs of the anti-immature sera against purified antigens, since these latter antigens essentially react with anti-peptide antibodies (hence, increasing the specificity and sensitivity of the serology). The N-terminus of the 260 kDa antigen shows 80-90% similarity with the hemoglobin alpha-chain of many mammals. The antigens of O. moubata are proteins that are very different from one another and are, therefore, readily purified by ion exchange chromatography. The 20A1 antigen appears to be the most immunogenic and is recognized equally by anti-immature and anti-adult sera. This antigen does not give false positive reactions with the negative control sera analyzed and its N-terminus region shares 46.2% homology with the alpha-chain of the C3 component of rabbit complement.     

Immunohistochemical examination of PDGF-AB, TGF-beta and their receptors in the hemocytes of a tick, Ornithodoros moubata (Acari: Argasidae)

Growth factors, Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor (TGF)-beta, were demonstrated in vertebrate and invertebrate immmunocytes. It is generally known that the growth factors are important in various biological processes, such as the regulation of cell differentiation, development and wound healing. In the present study, the presence of TGF-beta1 and PDGF-receptor-alpha in plasmatocytes and PDGF-AB in granulocytes of a soft tick, Ornithodoros moubata, was confirmed immunohistochemically. The tick midgut might be damaged by intracellular digestion and penetration of protozoa. Therefore, it is considered that PDGF from granulocytes may affect the PDGF-receptor-alpha in plasmatocytes and TGF-beta from plasmatocytes may function to repair the midgut. The results obtained here add to the elucidation of the functions of tick hemocytes.     

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.     

Development, characterization, and lethal effect of monoclonal antibodies against hemocytes in an adult female tick, Ornithodoros moubata (Acari: Argasidae)

In the present study, 19 monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established, and the reactivity of the hemocytes to these mAbs was examined by an indirect fluorescent antibody test (IFAT), Western blot and immunoprecipitation analyses. It was shown that the reactivities of the hemocytes to the mAbs varied among morphologically similar hemocyte types, and most mAbs produced in the present study showed the multiple band reactivity. However, the presence of shared epitopes among peptide subunits of the same protein or entirely different proteins are not common, so their reactivity could not be explained in detail. These results suggest that there are morphologically similar but functionally differentiated hemocytes. Therefore, in addition to morphological classification, the molecular-based classification of the hemocytes is also required. In order to assess the lethal effect of blood meal containing each mAb, artificial feeding was performed. The OmHC 31 showed the strongest lethal effect on adult female O. moubata. In conclusion, anti-hemocyte mAbs produced in this study are useful not only for the immunological classification of hemocytes but also for the immunological control of the tick.     

Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus

A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.     

IV. Demonstration of the Viral Antigen by Means of Immunofluorescence

African swine fever immunofluorescent conjugates were prepared in swine and used successfully in the demonstration of viral antigen in frozen tissue sections and in inoculated tissue culture cells. Cross reactivity was observed with the six strains used in the inoculation of swine. The high antibody content of the serum of immune swine did not interfere with demonstration of the antigen in frozen tissue sections of certain of their organs. The localisation and extent of antigen varied with the stage of infection. The virus was demonstrated in spleen and other organs as early as after one day of pyrexia and until after death of the animal. A pool of hog cholera and African swine fever conjugates stained with dyes of different colours was used in the localisation of respective antigens in experimental mixed infection.     

Research Progress on Epidemiology of African Swine Fever

African swine fever (ASF) is mainly occurred in Africa.It invades into Europe and America by chance,then invades in to Eastern Europe and the Caucasus area.And it has not been controlled until now.African swine fever virus (ASFV) can spread by soft tick vectors and affect wild and domestic pigs.The ability of the virus to survive within a particular ecosystem is defined by the ecology of its wild host populations and the characteristics of livestock production systems.African swine fever is viral disease of domestic and wild pigs which leads high mortality and causes great economic losses due to absence of available vaccine and treatment.Apart from prevention and culling,there are no other control measures.Prevention and control of the infection require good understanding of its epidemiology,so that targeted measures can be carried out.     

某养殖场的病死野猪ASFV实时荧光PCR检测

为了进一步做好非洲猪瘟疫情防控工作,研究用实时荧光PCR法对武威市某养殖场病死野猪的猪耳朵和猪鼻拭子样品进行了非洲猪瘟病毒检测。结果显示该养殖场病死猪耳样品和猪鼻拭子样品均无Ct值,线形为直线,表明所有被检测样品均为ASFV抗原阴性。研究为严防非洲猪瘟进入威武市奠定了坚实的技术储备,也为威武市非洲猪瘟疫情防控提供了一定参考。