Is Ornithodoros erraticus able to transmit the Georgia2007/1 African Swine Fever virus isolate to domestic pigs?

Objective: African swine fever, one of the most devastating diseases affecting wild and domestic swine, is due to a large DNA virus, only member of the Asfarviridae family. After ASF introduction in Georgia in 2007, the disease became endemic in the Caucasian region of the Russian Federation and spread towards the Western regions of Europe entering in EU Members States at the beginning of 2014. As no vaccine or antiviral are available to fight against this infection, the only tools to control it are preventive measures based on early detection and actual knowledge of the epidemiological risks. In African sub-Saharan countries, ASF persistence is described to be related to different and complex epidemiological scenarii involving domestic and wild suids and soft tick vectors of the genus Ornithodoros. In EU, one species of Ornithodoros, O. erraticus, is known to be able to maintain and/or transmit some ASFV isolates classified in the genotype I. Recently, the Pirbright Institute also demonstrated that O. erraticus was able to amplify the Georgia2007/1 ASFV (genotype II), at least during 3 months. The objective of the current study was to evaluate the in- vitro and in-vivo transmissibility of the Georgia 2007/1 ASFV by infected O. erraticus ticks. Methods: The Georgia 2007/1 ASFV strain belonging to the genotype II, kindly provided by L. Dixon (OIE reference lab), was grown on porcine pulmonary alveolar macrophages to the titre of 106to 107 HAD50 , then diluted 100-fold into pig blood for tick infection or 1000-fold in medium for intradermal inoculation to pigs. Ticks were captured in Portugal by F. Boinas and mass reared at CIRAD for one year and a half to obtain a stable and mature population. During this period, several techniques of artificial feeding were tested to optimize the method. In December 2014, 60 adults or last nymphal stages -group A- coming both from field and 1st generation laboratory were artificially engorged on pig blood supplemented with Georgia 2007/1 at a final titre of 104.5 HAD50/mL blood. Two other groups of ASFV-free ticks –group B with 60 individuals and group C with 30 individuals- were reared to be used for a second infection directly on infected pigs (group B) and as control group (group C), respectively. Moreover to confirm the possibility to infect ticks through artificial blood meal, another group of 10 ticks was also engorged and tested for virus multiplication three months later. Fifteen other females were also infected and secondarily engorged on ASFV-free pig blood to test in-vitro transmission through virus isolation on second blood meal. Considering that it is difficult to obtain ASFV titres with in-vitro cultivation as high as in infected pigs developing ASFV clinical signs, it seems important to compare ASFV transmissibility between ticks artificially infected in laboratory and ticks directly infected on ASFV-infected pigs and conclude on possible dose effect. In March 2015, 18 Large-White pigs obtained from a high sanitary level field herd were distributed to 4 groups at Anses-Ploufragan high security facilities. Two negative control groups of 3 pigs were either intra-dermally inoculated with MEM or bitten by group C of 30 healthy ticks. One group of 6 pigs was intra-dermally inoculated with 103 HAD50 ASFV while the last group of 6 pigs was bitten by group A of 60 ticks previously infected through artificial blood meal and dispatched in 10 ticks/pig. Pots of 10 ticks were placed on one ear held there with adhesive tape, then removed after 3 hours. After removal, ticks were numbered in two batches: engorged and unengorged ticks. Finally, as soon as the 6 pigs intra-dermally inoculated with ASFV showed fever and high viremiae, group B of 60 ASFV-free ticks were proposed to engorge on their opposite ear. These ticks would be proposed to secondarily engorge on membrane feeding or healthy pigs three months later. Post tick feeding or intradermal inoculation, clinical examination and rectal temperatures were recorded daily, until the animals were euthanized or for a minimum period of 18 days. Except on D1 pi, serum and EDTA blood samples were daily collected from all the pigs during the first week pi, then twice a week during the 2 following weeks, and at the day of euthanasia for virological and serological assays. Organ samples were collected during necropsy. The animal experiment protocol was approved by the French national ethics committee ComEth Anses/ENVA/UPEC (10/03/15-9). Results: Ten ticks from the original batch of ticks that were artificially fed on infectious blood were tested by virus titration. Out of them, 8 were positive with a titre ranging from 102 to 104.2 HAD50/tick and 2 ticks clearly amplified the virus regarding the estimated amount of virus originally ingested (minimum of 1 log superior). After feeding on pigs, the mean level of engorged ticks was of 62%, whatever the group of pigs. The experiment, currently running, confirmed the high virulence of the Georgia strain as all the 6 intra-dermally inoculated pigs displayed typical symptoms of ASF including lost of appetite and hyperthermia from D3 pi. The 6 pigs were euthanized from D5 to D7. The group of the 6 pigs bitten by the infected ticks was still healthy at 18 days post feeding, as well as the two negative control groups. However, among the 15 female ticks secondarily engorged on ASFV-free pig blood, no heamadsorption effect was observed after two passages on alveolar macrophage culture using blood-meal leftovers. Further investigations are needed to confirm the presence of ASF Virus. The final experimental infection of pigs through tick bite using ticks previously engorged on viremic pigs should allow concluding on the ability of O. erraticus to transmit Georgia2007/1 and a possible dose effect on this transmissibility. The results will be presented and discuss during the symposium. Conclusion: The objective of this study was to experimentally assess the ability of the European O. erraticus tick to transmit the Georgia 2007/1 ASFV currently circulating in North-Eastern EU. First results showed that ticks artificially infected in laboratory did not induce ASF clinical signs in pigs by biting. However, virus titration in ticks seems to show that the virus is present in the arthropod. Further in-vitro and in-vivo investigations are running to explore the hypothesis of a dose effect. The expected results should clarify the potential epidemiological role of O. erraticus ticks in transmission and re-emergence of ASFV in the field, in case of the spread of current active foci from North-Eastern EU. (Résumé d'auteur