African horse sickness Epidemiology: Culicoides spp. diversity, vectors and overwintering of the virus in Eastern Cape Province, South Africa

Abstract

African horse sickness (AHS) is a devastating, non-contagious viral disease of equids. However, zebra are resistant to the disease. Previously it was accepted that outbreaks of AHS would start in the northern parts of South Africa and gradually move southwards as the season progressed. This stemmed from the belief that the large population of zebras in the Kruger National Park, acted as the source of the virus. In outbreaks that have occurred over the past 25 years, it has become evident that this was not true, as outbreaks would persist for two or more seasons in the south of the country, with the same AHS serotype causing the first outbreak being the only one recorded in that area in subsequent years. Therefore, it is possible that the virus overwinters in the area. However, the overwintering mechanism of the African horse sickness virus (AHSV) is not fully understood. It has been suggested that the AHSV overwinters in adult Culicoides populations, at low levels of viremia in natural hosts, or in other vertebrate or invertebrate hosts such as dogs and ticks, respectively. The aim of this study was to investigate how the AHSV overwinters in the study area, and the role dogs and ticks play in this process and to determine the diversity for both Culicoides and tick species in the study area. This study was undertaken in the Sarah Baartman district in the Eastern Cape Province of South Africa. A total of 28 sites were recruited for collection of Culicoides and tick species, and blood samples from dogs. A 220 V down draught-UV light trap was used to collect Culicoides specimens, while ticks were collected directly from live hosts. Blood samples were drawn from dogs using the cephalic vein. All these samples were sent to the Agricultural Research Council – Onderstepoort Veterinary Research (ARC-OVR) for analysis. Over the survey period 918 collections of Culicoides were made, and 44,850 Culicoides specimens, comprising of 49 Culicoides species were recovered. A total of 1,260 tick specimens comprising of 10 species from three genera were collected and identified during the survey. Over a period of one year, blood samples were collected from 100 dogs. Each dog was sampled four times, once every season. Blood samples were analysed using an AHS indirect ELISA to test for the presence of IgG antibodies. Culicoides and tick samples were first tested for the presence of AHSV using an inhouse PCR. All samples that tested positive on the inhouse PCR were subjected to the WOAH (accredited and validated) test. Only samples that tested positive with the WOAH test were considered AHSV positive. While five Culicoides pools tested positive with the inhouse PCR, only two pools of these pools tested positive for the virus on the WOAH PCR test. Furthermore, of the two pools that tested positive: one was from Site 1 and consisted of two specimens of C. bolitinos and the other pool was from Site 7 and consisted of six specimens of C. tuttifrutti. This study provides an insight into the diversity of both Culicoides and tick species in the study area. Findings reported here demonstrated that dogs are not preferred hosts for Culicoides species given that none of the dogs tested positive for AHSV antibodies. Based on the results from this study, only Culicoides species have the potential to play a role in the overwintering of AHSV in the area. However, additional tick species and other possible vertebrate hosts should also be investigated. Further research is needed to investigate the role of C. tuttifrutti and other blood feeding flies in the transmission of AHSV and other orbiviruses.

Afrika-perdesiekte (APS) is ’n verwoestende, nie-aansteeklike virale siekte van perdagtiges. Sebras is egter bestand teen die siekte. Voorheen is aanvaar dat uitbrekings van APS in die noordelike dele van Suid-Afrika sou begin en geleidelik suidwaarts sou beweeg soos die seisoen vorder. Dit het gespruit uit die oortuiging dat die groot bevolking sebras in die Krugerwildtuin die bron van die virus was. In uitbrekings die afgelope 25 jaar, het dit duidelik geword dat dit nie waar is nie, aangesien uitbrekings vir twee of meer seisoene in die suide van die land sou voortduur, met die APS-serotipe wat die eerste uitbreking veroorsaak het die enigste een wat in die daaropvolgende jare aangeteken is. Daarom is dit moontlik dat die virus in die gebied oorwinter. Daar is egter nie voldoende begrip van die oorwinteringsmeganisme van die Afrika-perdesiektevirus (APSV) nie. Daar is voorgestel dat die APSV oorwinter in volwasse Culicoides-bevolkings, teen lae vlakke van viremie in natuurlike gashere, of in ander werweldiere of ongewerwelde diere soos honde en bosluise, onderskeidelik. Die doel van hierdie studie was om te ondersoek hoe die APSV in die studiegebied oorwinter, die rol wat honde en bosluise in hierdie proses speel, en om die diversiteit van beide Culicoides- en bosluis spesies in die studiegebied te bepaal. Hierdie studie is in die Sarah Baartman distrik in die Oos-Kaap provinsie van Suid-Afrika onderneem. Altesaam 28 terreine is besoek vir die insameling van Culicoides- en bosluis spesies, en vir bloedmonsters van honde. ’n 220 V-intreklug-UV-ligval is gebruik om die Culicoides-monsters te versamel terwyl bosluise direk van lewende gashere ingesamel is. Bloedmonsters is uit die kefaliese aar by honde getrek. Al hierdie monsters is na die Landbounavorsingsraad-Onderstepoort Veterinêre Navorsing (LNR-OVN) gestuur vir analise. Tydens die opname tydperk is 918 insamelings van Culicoides gemaak, en 44,850 Culicoides-monsters bestaande uit 49 Culicoides spesies is opgespoor. ’n Totaal van 1,260 bosluis-monsters bestaande uit 10 spesies uit drie genera is tydens die opname ingesamel en geïdentifiseer. Tydens ’n tydperk van een jaar is bloedmonsters by 100 honde ingesamel. Bloedmonsters is vier keer, een keer per seisoen, by elke hond ingesamel. Bloedmonsters is ontleed met behulp van ’n APS indirekte ensiemgekoppelde immunosorbenttoets (ELISA) vir die teenwoordigheid van IgG-teenliggame. Culicoides- en bosluismonsters is eers getoets vir die teenwoordigheid van APSV met behulp van ’n interne PKR. Alle monsters wat positief getoets het met die interne PKR is aan die Wêreld organisasie vir Dieregesondheid (WOAH) se geakkrediteerde en geldige toets onderwerp. Slegs monsters wat positief getoets het met die WOAH-toets is as APSV-positief beskou. Alhoewel vyf Culicoides-poele positief getoets het met die interne Polimerase kettingreaksie (PKR), het slegs twee poele van hierdie poele positief getoets vir hierdie virus met die WOAH-PKR-toets. Verder, van die twee poele wat positief getoets het, was een van Terrein 1 en het bestaan uit twee monsters van C. bolitinos, en die ander poel was van Terrein 7 en het bestaan uit ses monsters van C. tuttifrutti. Hierdie studie bied insig in die diversiteit van beide Culicoides- en bosluis spesies in die studiegebied. Die bevindinge wat hier gerapporteer is, het getoon dat honde nie voorkeur gashere vir Culicoides-spesies is nie, aangesien geen honde positief getoets het vir APSV-teenliggame nie. Op grond van die resultate van hierdie studie het slegs Culicoides spesies die potensiaal om ’n rol te speel in die oorwintering van APSV in die gebied. Addisionele bosluis spesies en ander moontlike gewerwelde gashere moet egter ook ondersoek word. Verdere navorsing is nodig om die rol van C. tuttifrutti en ander bloed voedende vlieë in die oordrag van APSV en ander orbivirusse te ondersoek.

Isifo samahashe iAfrican horse sickness (iAHS) sisifo esitshabalalisayo, esingosuleliyo. Nangona kunjalo, amaqwarhashe ayaxhathisa kwesi sifo. Ngaphambili kwakusamkelwa ukuba uqhambuko lweAHS luza kuqala kwiindawo ezisemantla oMzantsi Afrika luze ngokuthe chu lunwenwe ukuya emazantsi ngokuhamba kwexesha lonyaka. Oku kubangelwa yinkolelo yokuba uninzi lwamaqwarhashe eKruger National Park, lusebenza njengemvelaphi yale ntsholongwane. Kuqhambuko oluye lwenzeka kule minyaka ingama 25 adlulileyo, kuye kwacaca ukuba oku bekungeyonyani, nanjengoko ukuqhambuka bekuye kuqhubeke kangangezihlandlo ezibini okanye ngaphezulu ngexesha lonyaka kumazantsi eli lizwe, kunye neAHS efanayo ebangele ukuqhambuka kokuqala ibekuphela kwako ekubhalwe phantsi kule ndawo kwiminyaka elandelayo. Ngoko ke, kunokwenzeka ukuba le ntsholongwane inwenwa ubusika bonke kwindawo leyo. Nangona kunjalo, indlela yokunwenwa ubusika bonke kwentsholongwane yokugula kwamahashe iAfrika horse sickness virus (iAHSV) ayiqondwa ngokupheleleyo. Kuye kwacetyiswa ukuba iAHSV inwenwa ubusika bonke kwiimbuzane (Culicoides) ezindala, kumanqanaba aphantsi kokubakho kweentsholongwane egazini kumakhaya endalo, okanye kwamanye amakhaya ezilwanyane ezinethambo lomqolo okanye izilwanyana ezingenathambo lomqolo ezifana nezinja kunye namakhalane, ngokulandelanayo. Olu phando lwenziwe kwisithili iSarah Baartman kwiPhondo leMpuma Koloni eMzantsi Afrika. Kukhangelwe iindawo ezingama28 zizonke ukuze kuqokelelwe iintlobo zeembuzane, namakhalane, kunye neesampuli zegazi ezinjeni. Kusetyenziswe isibane sokuthiyisela/sokubamba izinambuzane i220 V down draught-UV ukuqokelela iisampuli zeembuzane, ngelixa amakhalane aqokelelwa ngokuthe ngqo kwizilwanyane eziphilayo. Iisampuli zegazi zitsalwe kwizinja kusetyenziswa umthambo osemkhonweni. Zonke ezi sampuli ziye zathunyelwa kwiAgricultural Research Council – Onderstepoort Veterinary Research (iARC-OVR) ukuze zixilongwe. Ngexesha lophando kwenziwa ingqokelela yeembuzane ezingama 918, kunye neesampuli zeembuzane ezingama 44,850, ezibandakanya iintlobo zeembuzane ezingama49 ezafunyanwayo. Zizonke ziisampuli zamakhalane ali 1,260 eziquka iintlobo ezili10 ezivela kumaqela amathathu afanayo ezithe zaqokelelwa zaze zachongwa ngexesha lovavanyo. Kwisithuba esingangonyaka, iisampuli zegazi zaqokelelwa kwizinja ezili100. Kwathathwa iisampuli zegazi kane kwinja nganye, kanye ngexesha lonyaka. Iisampuli zegazi zaxilongwa kusetyenziswa iAHS indirect ELISA ukuvavanya ubukho bezithintelintsholongwane zeIgG. Iisampuli zeembuzane nezamakhalane zavavanywa kuqala ukujonga ubukho beAHSV kusetyenziswa iPCR yangaphakathi. Zonke iisampuli eziye zafunyaniswa zinentsholongwane kwiPCR yangaphakathi zifakwe kuvavanyo lweWOAH (oluvunyiweyo noluqinisekisiweyo). Ziisampuli kuphela eziye zafunyaniswa zinentsholongwane ngovavanyo lweWOAH eziye zathathwa ngokuba zineAHSV. Ngelixa amagcuntswana amahlanu eembuzane afunyaniswa enentsholongwane ngePCR yangaphakathi, ngamagcuntswana amabini kuphela kula magcuntswana afunyaniswe enentsholongwane kuvavanyo lweWOAH PCR. Ngaphaya koko, kumagcuntswana amabini afunyaniswe ukuba anayo le ntsholongwane: elinye belisuka kuSite 1 kwaye lineesampuli ezimbini ze C. bolitinos, elinye igcunstwana belisuka kuSite 7 kwaye lineesampuli ezintandathu ze C. tuttifrutti. Olu phando lunikeza ulwazi ngentlobo ntlobo zeembuzane kwakunye neentlobo ntlobo zamakhalane kwindawo uphando ebelubanjwe kuyo. Iziphumo ezixelwe apha zibonise ukuba izinja azikhethwa ziintlobo zeembuzane kuba akukho nanye kwizinja ezivavanyiweyo efunyaniswe inezithintelintsholongwane zeAHSV. Ngokusekelwe kwiziphumo zolu phando, ziintlobo zeembuzane kuphela ezinokuthi zidlale indima yokunwenwa ubusika bonke kweAHSV kwindawo. Nangona kunjalo, iintlobo zamakhalane ezongezelelweyo kunye nezinye izilwanyane ezinethambo lomqolo ezinokubakho kufuneka ziphandwe nazo. Uphando olongezelelweyo luyafuneka ukuphanda indima ye C. tuttifrutti nezinye izinambuzane ezifunxa igazi ekusasazeni iAHSV kunye nezinye iintsholongwane ezisuleleka ngenxa yezinambuzane (i-orbiviruses).

Lefu la Dipere la Afrika (AHS) ke lefu le sithabetsang, le sa tshwaetsaneng la kokwana-hloko ya mefuta e anyesang ya dipere. Leha ho le jwalo, diqwaha ha di be le lefu lena. Pele ho ne ho amohelwa hore ho qhoma ha AHS ho tla qala dikarolong tse ka Leboya la Afrika Borwa mme butle-butle le lebe dikarolong tse borwa ha nako ya selemo e ntse e tswela pele. Sena se ne se bakwa ke tumelo ya hore palo e ngata ya diqwaha tse Kruger National Park, e sebetsa e le mohlodi wa kokwana-hloko. Ho qhoma ho etsahetseng dilemong tse 25 tse fetileng, ho bonahetse hore sena e ne e se nnete, kaha mafu a sewa a ne a tla tswela pele ka dinako tse pedi kapa ho feta tsa selemo karolong e borwa ho naha, ka mokgwa wa ho bokella disele kapa dikokwana-hloko, o tshwanang wa AHS e bakang sewa sa pele e le sona feela se tlalehilweng sebakeng seo dilemong tse latelang. Ka hona, ho ka etsahala hore kokwana-hloko e phele nakong yohle ya mariha sebakeng seo. Leha ho le jwalo, ho phela nakong yohle ya mariha ha kokwana-hloko ya lefu la dipere tsa Afrika (AHSV) ha ho so utlwisiswe ka botlalo. Ho ile ha etswa tlhahiso ya hore AHSV e phela nakong yohle ya mariha ho di-Culicoide tse kgolo, ka maemo a tlase a boteng ba vaerase mading ho baamohedi ba tlhaho, kapa ho dihlopha tse ding tsa diphoofolo tse nang le mokokotlo kapa tse se nang mokokotlo tse kang dintja le diboseleise, ka ho latellana. Sepheo sa phuputso ena e ne e le ho batlisisa hore na AHSV e phela mariha oohle jwang sebakeng sa phuputso, le karolo eo dintja le diboseleise di e bapalang tshebetsong ena le ho fumana hore na mefuta ya di-Culicoide le diboseleise e na le mofuta ofe sebakeng sa phuputso. Phuputso ena e entswe seterekeng sa Sarah Baartman Porofenseng ya Kapa Botjhabela Afrika Borwa. Kakaretso ya dibaka tse 28 e ile ya ngodiswa bakeng sa pokello ya mefuta ya di-Culicoide le diboseleise, le disampole tsa madi a dintja. Sefi sa lebone sa 220 V down draught-UV se ile sa sebediswa ho bokella mehlala ya di-Culicoide, ha diboseleise di ne di bokellwa ka ho toba ho baamohedi ba phelang. Ho ile ha nkuwa disampole tsa madi ho dintja ho sebediswa mothapo wa cephalic. Disampole tsena kaofela di ile tsa romellwa Lekgotleng la Dipatlisiso tsa Temo - Onderstepoort Veterinary Research (ARC-OVR) bakeng sa tlhahlobo. Nakong ya phuputsi ho ile ha etswa dipokello tse 918 tsa di-Culicoide, mme mefuta e 44,850 ya di-Culicoides, e nang le mefuta e 49 ya di-Culicoides e ile ya fumanwa. Kakaretso ya mehlala ya diboseleise tse 1,260 tse nang le mefuta e 10 ho tswa melokong e meraro e ile ya bokellwa le ho tsejwa nakong ya dipatlisiso. Ka nako ya selemo se le seng, ho ile ha bokellwa disampole tsa madi ho dintja tse 100. Ntja ka nngwe e ile ya etswa disampole ka makgetlo a mane, hanngwe ka nako ya selemo. Disampole tsa madi di ile tsa hlahlojwa ho sebediswa ELISA e sa tobang ya AHS ho etsa tlhahlobo ya boteng ba disele tse sireletsang mmele mafung tsa IgG. Di-Culicoide le disampole tsa dibosoleise di ile tsa lekwa pele bakeng sa boteng ba AHSV ho sebediswa PCR e teng. Disampole tsohle tse fumanweng di na le AHSV ho PCR e teng di ile tsa etswa tlhahlobo ya WOAH (e amohelehang le e tiisitsweng). Ke feela disampole tse fumanweng di na le AHSV ka tlhahlobo ya WOAH di ileng tsa nkuwa di na le AHSV. Le hoja matamo a mahlano a di-Culicoide a ile a fumanwa a e na le PCR ya kahare, ke matamo a mabedi feela a matamo ana a ileng a fumanwa a e na le kokwana-hloko tekong ya WOAH PCR. Ho feta moo, ho matamo a mabedi a ileng a fumanwa a e na le AHSV: le leng le ne le tswa Setsing sa 1 mme le ne le na le mehlala e mmedi ya C. bolitinos le letamo le leng le ne le tswa Setsing sa 7 mme le ne le na le mehlala e tsheletseng ya C. tuttifrutti. Phuputso ena e fana ka temohisiso ka mefuta-futa ya mefuta ya di-Culicoide le diboseleise sebakeng sa phuputso. Diphumano tse tlalehilweng mona di bontshitse hore dintja ha di ratehe bakeng sa mefuta ya di-Culicoid kaha ha ho le e nngwe ya dintja tse fumanweng di na le disele tse sireletsang mmeleng mafung tsa AHSV. Ho ipapisitswe le diphetho ho tswa phuputsong ena, ke mefuta ya di-Culicoide feela e nang le monyetla wa ho bapala karolo ya ho phela mariha oohle wa AHSV sebakeng seo. Leha ho le jwalo, mefuta e meng ya diboseleise le diphoofolo tse ding tse nang le lesapo la mokokotlo le tsona di lokela ho etswa dipatlisiso. Ho hlokahala dipatlisiso tse ding ho batlisisa karolo ya C. tuttifrutti le ditshintsi tse ding tse fepang madi phetisong ya AHSV le dikokwana-hloko tse ding tsa orbivirus.