SE2610: Effect of Temperature and Regional Origin on the Vector Capacity of British And
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8. Project report to DEFRA.
SE2610: Effect of Temperature and Regional Origin on the Vector Capacity of British and European Culicoides spp for bluetongue virus.
Initial Scientific objectives:
1. To measure the vector competence, for BTV, of populations of C. obsoletus, C. pulicaris and other potential vector species complexes of Culicoides originating from widely varying locations in the UK in order to gain a true estimate of the risk of BTV transmission in the country.
2. To identify, from the findings of objective 1, the locations of any Culicoides populations with high levels of competence so that areas at high risk of BTV transmission can be demarcated and the appropriate disease control measures designed in advance of any disease incursion.
3. To investigate the effects of ambient temperature in modifying vector competence for BTV, including the possibility of facilitating transovarial transmission of the virus.
4. To investigate the bioclimatic envelope of C. imicola, the major European BTV vector, to assess the likelihood of this species surviving as a population in the UK should it gain entrance to this country.
Extent to which Scientific Objectives have been fulfilled:
1. Fulfilled. A novel means of feeding wild-caught Culicoides species a BTV-spiked blood meal was devised following failure of standard feeding techniques. Using this method, vector competence for BTV was successfully measured at 12 sites across the UK over the three years of the project. Species assessed in this way included the C. obsoletus complex, the C. pulicaris complex and C. impunctatus. Variable proportions of all these groups were shown to be able to become infected by, and replicate the virus, some to levels confirming that transmission could take place.
2. Fulfilled. C. obsoletus complex midges from two of ten sites examined for that species showed consistently high levels of vector competence for BTV. C. pulicaris complex midges were also shown to possess a high degree of competence at one site in the survey. These geographic differences in competence did not appear to be strictly demarcated.
3. Partially fulfilled. No evidence was found to support transovarial transmission of BTV in laboratory experiments with the known BTV vector C. sonorensis. Only limited data regarding variations in the duration of the extrinsic incubation period (i.e. time from virus ingestion to transmission) at different temperatures was produced due to time constraints. However, this work did confirm that the topotype of BTV 9 from the most northern location in Europe to where BTV had penetrated replicated in vector midges more quickly than other topotypes of this serotype. Further work is planned in this area as part of an additional BBSRC/Defra project.
4. Fulfilled. C. imicola is unlikely to become established permanently at present in the UK due to its bioclimatic requirements not matching those currently occurring in this country. The likelihood of transmission of BTV by Palaearctic Culicoides already present in the UK, following introduction via movement of infected ruminants or midges, however, has not been assessed and will form the main body of the BBSRC/DEFRA funded project mentioned above.
Objective 1.
Section 1: Testing of membrane-based feeding methods with field-caught Palaearctic Culicoides species and quantitative validation of a novel feeding technique.
Methods Several different membrane-based methods of feeding Culicoides to test their susceptibility to infection with BTV were initially trialled for use in the project using C. obsoletus complex midges collected from a site local to IAH Pirbright (see Mellor, 1971; Venter et al., 1991 for details of feeding methods). None of the membrane systems used produced the rates of feeding required to successfully carry out objectives 1 and 2, with blood fed midges rarely comprising more than 1% of the total number introduced. This was despite the fact that the techniques used had proved to be efficient and reliable with closely related Culicoides species in other countries in numerous related studies. A wide range of factors that could enhance feeding rates were then investigated in an attempt to enhance these extremely low rates, including offering a variety of membrane types (nescofilm, chickskin of varying ages, parafilm and silicone-based film: Davis et. al., 1981) , varying the density of Culicoides when feeding (Blackwell et al., 1996), storing individuals prior to feeding under different conditions (Venter et. al., 1991), starving individuals during storage and varying the time that midges were left in the light trap prior to collection - with no enhancement observed. An alternative method was therefore devised whereby midges were offered the opportunity to feed on a blood/virus mixture via cotton wool “pads” positioned on top of netted pillboxes in which C. obsoletus complex midges had been placed. In this case up to 40% of the C. obsoletus complex midges would take a blood-meal with the proportion of midges feeding decreasing over time following removal from the light trap. At approximately 4h post-removal feeding rates were <1% of the midges introduced. As this was a novel method of feeding Culicoides, however, validation in comparison to the other traditional membrane-based techniques was required which was extremely difficult using the C. obsoletus complex midges with their low feeding rates on membrane based systems. Initially attempts to carry out this comparison focussed upon colony C. sonorensis, but it was found that this species would not ingest blood meal from a cotton wool pad under any conditions. It was found, however, that South African populations of C. imicola and C. bolitinos, (the former of which is the major vector of BTV in Europe and Africa, and the latter, one of the most susceptible Culicoides to BTV infection), would both feed through both membranes (using the method described by Venter et al., 1991), and on pads, and therefore provided an ideal means of quantitatively comparing the two techniques to allow pad-feeding to be used meaningfully with the closely related C. obsoletus complex in the UK. To carry out the comparison of feeding methods, C. imicola were collected using down-draught, 220V light-traps equipped with 8W UV-light tubes from at the ARC-OVI, Onderstepoort (2529’S, 2811’E; 1 219m a.s.l.) and C. bolitinos from Koeberg Farm near Clarens (2832’S, 2825’E; 1 631m a.s.l.) where it predominates. Down-draught 240V suction light-traps equipped with 8W UV-light tubes were used to catch insects overnight at these sites into 500ml plastic beakers, (partially filled with damp paper towel to protect trapped Culicoides from the down-draught of the trap and the severest effects of desiccation). These were removed at dawn, sealed, and transported to OVI for attempted oral infection. BTV’s used in experiments were obtained from the OIE Reference Laboratory for bluetongue at the ARC-OVI, South Africa (serotypes 1 and 5), and from IAH Pirbright (serotype 9) (Table 1).
Table 1: Titre and passage history of BTV strains used.
BTV Origin and strain Passage History Titre log10TCID50/ml serotype 1 RSA 1958 (Biggarsberg) 501E2,3P3,5BHK 6.1 5 RSA 1953 (Mossop) 50E,2BHK,3P,7BHK 6.6 9 Kosovo 2001 2E,3BHK 7.0
1 - Number of passages 2 - Embryonated hens eggs 3 - Plaque selection in green monkey kidney cells (Vero cells) 4 - Production of virus stocks in baby hamster kidney cells (BHK-21 cells)
Field collected Culicoides were fed in batches of 250-300 for 30-45 min on defibrinated sheep blood containing one of three serotypes of BTV through a one-day-old chicken-skin membrane as described by Venter et al., (1991; 1998). In parallel, midges were also fed, immediately post-collection, on 2-3cm2 cotton wool pads saturated in blood/virus mixture. Post-feeding, Culicoides were chilled and replete females separated and maintained in 250ml un-waxed paper cups for 10 days at 23.5ºC and 50-70% relative humidity. During incubation, 10% (w/v) sucrose solution containing antibiotics (500IU penicillin, 500µg streptomycin and 1.25µg per ml of fungizone) was made available via cotton wool pads. Culicoides surviving incubation were chilled and sorted into species before being stored individually in 1.5ml microfuge tubes at -70ºC prior to virus isolation. Individual midges were placed in autoclaved 1.5ml Eppendorf tubes containing 100μL of chilled Glasgow Minimum Essential Medium (MEM) with 0.6% antibiotics (2.0g/ml Fungizone 1000 IU/ml Penicillin, 50mg/ml Neomycin and 1000 IU/ml Polymyxin). They were then homogenised using autoclaved, motor driven, polypropylene pestles. Nine hundred μL of MEM was then added to each sample and the tubes centrifuged at 12000g for five minutes. Two hundred μL of the resulting supernatant was then used to prepare a 1:10 diluted sample using MEM in an additional Eppendorf tube for each sample. Virus titrations were carried out in tissue culture grade, 96-well microtitre plates containing a monolayer of BHK-21 cells and 100μL MEM supplemented with 2% tryptose phosphate broth (Invitrogen) and antibiotics. 100μL of each sample and its 1:10 dilution was inoculated onto plates in four replicates, together with a positive control of the original virus used (at 10- 4 to 10-7 dilution), and a negative control of dilutant. Plates were then sealed and incubated in a CO2 incubator at 37°C for 7 days, with microscopic observation for cytopathic effect (CPE) carried out at 5 and 7 days post-inoculation. Where CPE was observed the original samples were diluted again with an equal volume (500μL) MEM and passed through syringe-end filters (0.2 μm pore: Minisart). The resulting filtrate was then re-tested in an identical fashion, to a maximum 10-4 dilution. A BTV specific antigen-capture ELISA (OIE, 1996), was used to confirm virus identity and presence in all wells exhibiting CPE. Titres of positive samples for all species were calculated using the method of Spearman and Karbër (Finnay, 1964). An antigen-capture ELISA (OIE, 1996) and the virus neutralization test (Venter et al., 1998) using BTV serotype-specific antisera, were used for identification of virus isolates. Fisher’s exact test was used to compare the two methods of feeding and also to compare rates of virus isolation in C. imicola and C. bolitinos midges. A two-tailed Mann-Whitney test was used to compare the geometric mean virus titre in infected midges. Additionally, feeding was carried out six times for membrane fed midges (with pools ranging from 250 to 400) and five times for midges fed on cotton wool pledglets (with pools ranging from 1 000 to 2 000). The weights of the fed midges from each method were then compared to those of control groups of unfed midges. The blood meal size was calculated from the difference in weight, taking account of the specific gravity of the whole blood (1.06).
Results Virus infected individuals were recovered from both feeding methods, irrespective of the serotypes of virus used and species of Culicoides examined. In all cases, virus recovery rates (i.e. infection rates) were lower in midges fed via cotton wool pads or pledglets than through membranes (Table 2). Both membrane feeding (P = 0.003) and cotton pledglet feeding methods (P = 0.001) recorded higher infection rates in C. bolitinos than in C. imicola for BTV 1. The infection rates of BTV 9 fed C. imicola were significantly higher than for BTV1 with both feeding methods (cotton pledglet - P = 0.033; membrane feeding - P < 0.001) but were similar to BTV 5 (cotton pledglet - P = 0.6193; membrane feeding - P = 0.054). Similarly, the C. imicola infection rates for BTV 5 were significantly higher than with BTV 1 as determined using cotton pads (P = 0.009) or membrane feeding (P = 0.001). Mean virus titre for midges infected with BTV 1 did not vary significantly according to the method used to feed the two species (C. imicola: P = 0.787; C. bolitinos: P = 0.5476), or between the two species using the same feeding method (cotton pad: P > 0.9999; membrane feeding: P = 0.3833).
Table 2: Virus isolation rates and titres in Culicoides midges fed using two different feeding methods and three serotypes of BTV. P = comparison between feeding methods using Fisher’s exact test.
BTV serotype 1 5 9
Species Cotton Membrane P Cotton Membrane P Cotton Membrane P Pad Pad Pad
C. imicola Positive BTV 4/5871 7/402 0.13 8/254 10/115 0.03 8/327 75/468 <0.001 isolations (%) (0.7) (1.7) (3.1) (8.7) (2.4) (16.0) Range in virus tires2 0.7-2.4 07-2.4 0.79 ------Mean titre (SD) 1.4 (0.72) 1.4 (0.73) C. bolitinos ------Positive BTV 6/104 3/13 0.06 isolations (%) (5.8) (23.1) Range in virus titres 0.7-2.4 1.1-2.4 0.55 ------Mean titre (SD) 1.4 (0.58) 1.6 (0.70)
1 = Number of individuals positive/ no of individuals tested 2 = Log10/TCID50/ml
The blood meal size calculated from pooled C. imicola fed via membranes ranged from 0.023-0.062µl with a mean size of 0.045µl in six trials. Using cotton pads the blood meal sizes ranged from 0.019-0.038µl with a mean of 0.03µl. Consequently, the mean blood meal size obtained when pledglet feeding was significantly smaller (by about 33%) than when imbibing blood through a membrane (P = 0.0476). Discussion Blood feeding Culicoides using the pad or pledglet feeding method produced infection rates that in comparison with membrane feeding methods and previously published data accurately reflected variations due to the serotype and titre of virus used, and also distinguished between a highly susceptible species (C. bolitinos) and one that was less susceptible (C. imicola) (Venter et al., 1998; Paweska et al., 2002). However, the overall infection rates were significantly lower in cotton pad fed midges probably due to the reduction in the size of the blood meal by about 30%. The data presented show that the size of the blood meal taken by C. imicola (which is similar in size to C. obsoletus), when using a membrane system was about 0.045µl. This is not an absolute value as it does not take account of the elimination of excess liquid during feeding (Fujisaki et al., 1987; Leprince et al., 1989), but it can be used as a relative measure in comparisons between feeding methods, and it also equated to the blood meal volume taken from the natural host by the similarly sized Australian BTV vector C. brevitarsis (Muller et al., 1982). It was clear that when using the cotton pad method to attempt to assess the competence of potential vectors a sufficient number of insects was required to compensate for a 3 to 5 fold reduction in infection rate as shown in the present study. That criterion accepted, however, the cotton pad method allowed the subsequent assessment of the competence of potential UK vectors species that had proved virtually impossible to blood feed via a membrane or on the natural host subsequent to their capture.
Objective 1: Section 2. Assessing the competence of UK Culicoides
Methods & Materials Collections of Culicoides midges were made from a total of twenty-four locations across the UK, twelve of which supported sufficiently large populations for use in susceptibility trials (Fig.1). Culicoides were collected as in Section 1 and fed using the cotton wool pad method described by Venter et al., 2005, using 4-5ml of 1:1 horse blood/virus suspension mixture. A BTV serotype 9 isolate from a northern epizootic in Kosovo, identified and typed at IAH Pirbright, UK was used to infect midges. This represented the most northerly sample of BTV available when studies were initiated (June, 2002), and came from an area where C. imicola has not been recorded despite Culicoides surveys. It had been subjected to one passage through eggs and three passages through BHK-21 cells, and was used at a titre of 106.5- 7.0 TCID50/ml when combined (1:1) with horse blood. Insects within the pillbox were lightly anaesthetised using CO2 and blood-fed individuals identified. Thirty C. obsoletus complex midges from a single site (Normandy) were removed following successful feeding and stored immediately at -80ºC, until required for virus isolation and ELISA - these were day 0 controls. The remaining blood-fed insects were transferred to a clean pill-box and incubated at 23-25ºC and 80-95% RH for 7-10 days, with 5% sucrose solution available via a cotton-wool pad placed on top of the pill-box. Following incubation surviving midges were identified and stored in 1.5ml Eppendorf tubes at -80ºC until required for virus isolation and ELISA which were carried out as for Section 1.
Results Twelve UK sites examined during the three year study yielded sufficient Culicoides to allow examination of BTV oral susceptibility. In total, 290 midges from three sites were screened for competence in 2002 (all C. obsoletus s.l.), 2341 from 8 sites in 2003 (1939 C. obsoletus s.l.; 152 C. pulicaris s.l.; 250 C. impunctatus) and 1595 from 9 sites in 2004 (1455 C. obsoletus s.l.; 140 C. impunctatus). C. obsoletus s.l. was by far the most abundant group at almost all the sites, with the exception of the Scottish samples, where C. impunctatus predominated. C. pulicaris s.l. was present at all sites in low numbers (usually <20) with the exception of the samples from Scotland. Oral susceptibility to infection with BTV9 in C. obsoletus s.l. midges fed using the pad method varied according to the population examined (Table 3). Relatively low susceptibility to infection (2% of individuals examined) was recorded in populations of this complex taken from sites at Newmarket, Chobham, Wye, Allemoor, Upper-Langford and Normandy. Higher levels of susceptibility were recorded at Heath Mill (3.4% in 2003; 2.2% in 2004), Elstead (4.1% in 2004), Voss Farm (6.0% in 2003; 6.6% in 2004) and Keele (6.7% in 2002; 7.4% in 2003; 6.7% in 2004).
Fig.1. Collection sites used during the study of vector competence.
Viral titres obtained from susceptible C. obsoletus s.l. across the sites at 7-10 days post ≤0.8-4.8 infection ranged from 10 log10TCID50/individual (Table 3). Mean viral titres for the sites ≤0.8 2.5 examined ranged from 10 to 10 log10TCID50/individual. In total, 46 individuals replicated 0.8-1.9 2.0-2.9 3.0-3.9 the virus to 10 log10TCID50/individual, 56 to 10 log10TCID50/individual, 13 to 10 4.0-4.8 log10TCID50/individual and 2 to 10 log10TCID50/individual (Table 3). No positive virus isolations were made from any of the 30 C. obsoletus s.l. individuals frozen immediately post-feeding from Normandy, indicating low volume blood meal ingestion. The population of C. pulicaris s.l. examined from Keele in 2003 was the most highly susceptible examined during the study (13.0% susceptible to infection; Table 4). In contrast, C. impunctatus from Kingussie in 2003 and Ormsary in 2004 were among the least susceptible of the populations examined (0.4% and 0% susceptible respectively: Table 4).
Table 3. Susceptibility to infection with BTV-9 of Culicoides obsoletus s.l. populations collected at 10 sites across the UK and their subsequent levels of viral replication. Viral titre in susceptible midges % Infection Log10TCID50/midge Site name Location Year Rate (n) 10 <0.8-1.9 10 2.0-2.9 10 3.0-3.9 10 4.0-4.8 Mean
2002 6.7 (75) 3 1 1 0 101.4 5300'N Keele 2003 7.4 (502) 15 19 3 2 102.1 0216'W 2004 6.7 (194) 7 3 3 0 101.9 2002 ------5121'N Chobham 2003 1.0 (313) 1 1 1 0 102.1 0036'W 2004 0.6 (163) 1 0 0 0 10<0.8 2002 0.9 (111) 0 1 0 0 102.0 5115'N Normandy 2003 0.4 (261) 0 1 0 0 102.0 0040'W 2004 ------2002 ------51.11’N Elstead 2003 ------0042’W 2004 4.1 (195) 4 3 1 0 101.6 2002 ------5215'N Newmarket 2003 ------0028’E 2004 0 (236) - - - - - 2002 ------5117’N Wye 2003 ------0056’E 2004 0.5 (221) 1 0 0 0 101.8 2002 1.9 (104) 2 0 0 0 101.0 Upper 5121'N 2003 2.0 (298) 1 5 0 0 102.3 Langford 0247'W 2004 ------2002 ------5112'N Allemoor 2003 2.3 (260) 1 4 1 0 102.3 0249'W 2004 ------2002 ------5223'N Voss Farm 2003 6.0 (100) 0 6 0 0 102.5 0200'W 2004 6.6 (196) 4 7 2 0 102.2 2002 ------5223'N Heath Mill 2003 3.4 (205) 4 2 1 0 101.5 0200'W 2004 2.2 (223) 2 3 0 0 101.7
Table 4. Susceptibility to infection with BTV-9 of Culicoides impunctatus and C. pulicaris s.l. populations collected at 3 sites across the UK and their subsequent levels of viral replication. Viral titre in susceptible midges Species/ % Infection Log10TCID50/midge Location Year Site name Rate (n) 10 <0.8-1.9 10 2.0-2.9 10 3.0-3.9 10 4.0-4.8 Mean
1.3 C. impunctatus 5553’N 2003 0.4 (250) 1 - - - 10 / Ormsary 0537’W C. impunctatus 5657'N 2004 0 (140) - - - - - / Kingussie 0400'W C. pulicaris s.l. 5300'N 2003 13.0 (69) 5 1 3 - 101.8 /Keele 0217'W
Discussion This section of the project demonstrates that C. obsoletus s.l. in the UK are capable of becoming infected by, and can replicate a central European topotype of BTV. Significant geographic variation in oral susceptibility to infection was also detected in this group, ranging from 0.4% to 7.4% susceptibility among the sites examined. The highly susceptible populations of C. obsoletus discovered at the Keele and Voss Farm sites are of particular concern (along with the C. pulicaris s.l. from Keele) as their susceptibility to oral infection exceeds that recorded for the major old world vector, C. imicola, using the same feeding method and virus isolate during Section 1. Subsequently, the same C. imicola population was shown to have a membrane-based feeding susceptibility rate of 16% for the BTV9 used suggesting susceptibility in these Palaearctic species the susceptibility rate could be extremely high in comparison to what was previously thought (i.e. a membrane feeding equivalent up to almost 50%). While vector susceptibility to infection is not the sole factor in determining the probability of BTV incursion or spread, it is likely that these processes will be enhanced in areas of greater susceptibility. At present, little is known regarding the ability of the Culicoides infected during the study to successfully transmit the virus to new hosts. Virus was not detectable in the 30 C. obsoletus tested immediately following feeding (probably due to the small blood meal size and hence the low titre of virus ingested). Hence virus detected in Culicoides at 7-10 days post-feeding during this study must represent progeny virus that has replicated within the midge. Mere presence of virus in a potential vector at 7-10 days post infection does not, however, guarantee a fully disseminated infection and the ability to transmit. In an earlier study, it was deduced that full virus dissemination in C. sonorensis, a much larger midge, was 2.5 characterised by individuals developing greater than 10 TCID50 BTV 7 to 10 days after their infection (Fu et al., 1999). In the case of the smaller C. obsoletus and C. pulicaris complex midges this threshold is likely to be significantly lower, and further work is required to establish precisely what it is. Nevertheless, our results with UK populations of C. obsoletus 4.0 s.l., which include the detection of virus titres up to at least 10 TCID50/midge at 7-10 days post infection that transmission of BTV by at least a proportion of infected individuals, will be possible.
Objective 2.
Methods See Objective 1. Results Three sets of sites were used during the overall survey of susceptibility of C. obsoletus s.l. that were clustered in geographic location to allow investigation of competence at a local scale (<10 km between sites). These were termed the Herefordshire, Bristol and Surrey groups (see Fig. 1. for locations of sites). While the pair of sites examined at Bristol exhibited little variation in susceptibility to infection using the pad method, the sites at Surrey varied from 0.4% at Normandy in 2003 to 4.1% at Elstead in 2004, and those in Herefordshire from 2.2% at Heath Mill Farm in 2004 to 6.6% at Voss Farm in 2004.
Table 5. Variation in susceptibility to infection at a local geographic range (<10km between sites sampled).
Group Year Site % Susceptibility (n) 2002 2003 2004 Herefordshire Voss Farm 6.0 (100) 6.6 (196) Heath Mill 3.4 (205) 2.2 (223) Bristol Upper Langford 1.9 (104) 2.0 (298) Allemoor 2.3 (260) Surrey Chobham 1.0 (313) 0.6 (163) Normandy 0.9 (111) 0.4 (261) Elstead 4.1 (195)
Discussion The results presented in this section illustrate that variation occurs in oral susceptibility to infection with BTV 9 of C. obsoletus s.l. populations at a local geographic scale. At present, the underlying cause of this variation remains uncertain. Foremost among current hypotheses is that the highly susceptible population of C. obsoletus s.l. recorded at Keele may be comprised of a different mix of sibling species within the C. obsoletus complex than has been tested elsewhere. In the UK C. obsoletus s.l. comprises four species (C. obsoletus Meigen, C. dewulfi Goetghebuer, C. chiopterus Meigen, C. scoticus Downes & Kettle), and C. pulicaris s.l., two (C. pulicaris Linnaeus, C. punctatus Meigen). Female C. obsoletus s.l., are indistinguishable from one another and the males of this complex, which can be separated, are rarely caught at light (Delecolle, 1985; Campbell & Pelham-Clinton, 1960). In contrast, females of C. pulicaris s.l., can generally be separated through wing markings, but it has long been observed that a variable proportion of individuals overlap in diagnostic characters and so cannot be easily differentiated (Campbell & Pelham-Clinton, 1960; Lane, 1981). The possibility of gene-flow between C. pulicaris and C. punctatus has also received support, through examination of genetic markers, suggesting con-specific status (Ritchie et al., 2004). Whatever the reasons underlying vector susceptibility, the stability of infection rates at those sites examined on two or three consecutive years in the present study suggests that differences between sites are either heritable or that the environmental factors influencing vector competence have been stable over at least a three year period at least one of the sampled locations. It is therefore essential that methods are devised to differentiate reliably between females of both species complexes. Genetic markers have already been used successfully to separate species within other Culicoides species complexes (e.g. Linton et al., 2002; Dallas et al., 2003; Ritchie et al., 2004). The application of similar methodologies to the C. obsoletus and C. pulicaris complexes would allow direct linking of species identification with competence studies. The reliable identification of vector species and determination of their competence levels is of prime importance in making accurate BTV risk assessments and, at present, in locations where C. obsoletus s.l. and C. pulicaris s.l. are the primary vectors, such risk assessments are possible at the complex level only (Capela et al., 2003; Torina et al., 2004; Purse et al., 2004). These difficulties, together with the consistent and novel results regarding the variation in susceptibility of the C. obsoletus complex presented here, have led to the initiation of a new project run in collaboration between IAH Pirbright and Aberdeen University to examine ways of identifying the members of this and the C. pulicaris group through the use of molecular markers (DEFRA project number SE 4101).
Objective Three
Section 1. Determining the extrinsic incubation period of BTV in Culicoides sonorensis.
Methods & Results Initially, attempts were made to define the extrinsic incubation period (EIP) of BTV in its insect vector at a range of constant temperatures. Colony bred C. sonorensis (a known vector of BTV in the USA) were used to investigate this area as no indigenous UK vector species have been colonised to date. Several thousand midges were initially fed on the same BTV serotype 9 as used in objective 1. Fed midges were selected under light CO2 anaesthesia and incubated at 15, 20, 25 or 30C. At 24h intervals post-feeding, 50 Culicoides were removed for up to 10 days post-feeding at 30C and 14 days post-feeding at 15, 20 and 25C. Initially, whole midges were homogenised individually and tested for infection as for objective 1. This method relied upon the fact that a previous author had found that C. sonorensis with a fully disseminated infection (i.e. one that could lead to transmission of the virus to a new host) contained more than 2.5 log10 TCID50/ml of BTV. However, following the processing of infected midges for 15 and 25C it became clear that the time point at which full dissemination of virus occurred in the midge (vital for determining the EIP) was masked by the residual presence of virus still to be excreted from the original blood-meal (Fig.2).
100 25C
BTV BTV 90 15C
50
/ml /ml 80 15C:
TCID 70 10
log 60 >2.5 >2.5
ating ating 50
replic 40
ensis 30
sonor 20 C. C.
% % 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (d) post-feeding
Figure 2. The effect of temperature upon viral replication of BTV serotype 9 (Kosovo) in C. sonorensis midges (n=50 at each time point). This problem was exacerbated by the apparent low competence of C. sonorensis for the Kosovo serotype 9 virus isolate being used (Fig.2: approximately 10%). To overcome this problem, a second experiment was carried out, this time utilising decapitation and detection of virus replication in the head (the virus being known to replicate in several head organelles, most notably the cerebral ganglia). This had the benefit of allowing direct confirmation of the extrinsic incubation period by removing the confounding factor of un-excreted virus. The methodology was identical to the previous experiment with the exception that decapitation was carried out upon all individuals reaching the end of their respective time periods post- feeding.
25 30C: BTV 25C:
ining ining 20 20C: conta
heads heads 15 ensis
sonor
oides oides 10
Culic % % 5
0 1 2 3 4 5 6 7 8 9 10 11 12 Time (d) post-feeding
Figure 3. The effect of temperature upon the time of appearance of BTV serotype 9 (Kosovo) in C. sonorensis midges representing a fully disseminated infection (n=50 at each time point).
At 30C fully disseminated infections were present in some individuals after just three days. This is substantially more rapid than the EIP’s found in previous studies of BTV using different serotypes. It was also observed that the proportion of C. sonorensis developing a fully disseminated infection was highest at the highest constant temperature used (30C). Generally, only small amounts of virus were recovered from the heads that were examined
(<2 log10 TCID50/ml in all cases). In the case of midges stored at 25 and 20C, however, extremely low rates of virus recovery precluded further analysis, although it is worth noting the first appearance of the virus in the heads of individuals at five and six days respectively.
Discussion The accurate defining of the duration of the EIP for the Kosovan BTV-9 strain at different temperatures proved to be extremely difficult for a number of reasons. Firstly, initial work on this section of the project was driven by the need to produce consistent data in spite of the relatively low competence that the model species, C. sonorensis, had for this particular strain of BTV. This led to the use of large sample sizes in the two phases of experimentation (n=50 for each time period at each temperature; a total of 4000 C. sonorensis were fed and 2850 processed via BHK-21 cell monolayer virus isolation during the two trials). A pattern is discernable from the data of both trials, with evidence, especially in figure 3 showing that this topotype appears to replicate in the insect host more rapidly than other BTV-9 topotypes examined elsewhere. However, further work is necessary to confirm these studies and so this work area will be addressed in greater detail in a new BBSRC/DEFRA funded project,
Section 2. Investigation of the effect of temperature in facilitating transovarial transmission.
Methods To investigate transovarial transmission, almost 2000 colony bred C. sonorensis were fed upon a BTV 2 (USA strain)/ blood mixture and 1066 replete females were separated into pill boxes. Egg depository pots were included, which allowed egg-laying onto filter paper, and the insects incubated at 35◦C for 5 days (thermal stress having been shown to increase competence for BTV). The 724 surviving midges were given a 2nd blood-meal without virus and engorged females (226) separated and incubated as before. On days 2, 3 and 4 eggs were removed with the filter paper and stored at -80°C. A total of approximately 1600 eggs were collected with the majority being laid on days 3 (>1000) and 4 (>500). Culicoides eggs were then homogenised in pools according to day, and a Qiagen Rneasy Mini Kit was then used to extract RNA from the sample. An RT-PCR Qiagen onestep kit was then used with BTV specific primers for VP7 to amplify this region and allow detection of viral RNA in the samples. Following this, the surviving Culicoides sonorensis (187) were titrated on BHK cells and CPE was looked for on days 5 & 7 with confirmation using a BTV specific ELISA as in section 1.
Results Despite successful virus infection of positive controls and a large proportion of the adult midges testing positive for BTV infection (81%), none of the homogenised egg samples tested positive for BTV RNA when examined via PCR.
Discussion The relatively recent discovery of viral RNA in larvae of C. sonorensis in America (White et al., 2005), has revived interest in the possibility that BTV can be transovarially transmitted from infected adults to their progeny thus providing an effective overwintering mechanism via infected larvae and leading to disease outbreaks in the spring when the new generation of transovarially infected adults emerges. Our studies, however, illustrate that, despite a high proportion of females being found to be carrying a fully disseminated infection, no viral RNA was detectable in any of the eggs deriving from these females. While it is possible that either the RNA extraction procedure was inefficient, perhaps due to difficulties homogenising the eggs obtained, or that viral RNA was present in such small amounts as to be undetectable, these results appear to echo those of several previous authors who also failed to demonstrate the transovarial transmission of BTV through its Culicoides vectors. All of these possibilities, plus the attempted detection and/or isolation from progeny larvae and adults will be examined more thoroughly as part of a new BBSRC/DEFRA initiative Objective Four
Methods Data from a current EU project based at IAH and designed to predict risk of BTV incursion into Europe using satellite imagery was used to predict the likelihood of C. imicola s.s., the major vector of BTV in Europe, reaching and becoming established in the UK (see Tatem et al., 2003 for full review). During this study a model of C. imicola s.s. abundance was predicted for Europe derived from the observed abundances of this species at 87 sites in Portugal and combined with 1km resolution remotely sensed (RS) variables from the National Oceanic and Atmospheric Administration’s (NOAA) meteorological satellites over the period 1982-1994. This was then used to predict a northern limit to the establishment of C. imicola, given the conditions under which the vector is known to be present, and additionally, here, used to examine the likelihood of a shift in its northern limit. Additionally, changes in the temperature and precipitation between the 1990’s and 1980’s were quantified for Europe using monthly climate surfaces from the Climate research unit at the University of East Anglia, UK.
Results The predicted distribution of high density C. imicola populations produced from RS variables was found to correlate well with trapping data that had identified a northwards shift in the Northern distribution limit of C. imicola (Fig. 4a). Of the RS variables used, the Normalised Difference Vegetation Index (NDVI), a measure of radiation absorbed by chlorophyll during plant photosynthesis that is a correlate of soil moisture, vegetation biomass and productivity, was the single most important predictor of C. imicola abundance. The probable reason for this is that C. imicola breeds in wet but not saturated soils so that NDVI can be a good predictor of its breeding sites a) b)
Figure 4. Predictive map of areas of a) Southern Europe and b) the UK that correlate with remotely sensed variables determining C. imicola distribution (reproduced with permission from Tatem et al., 2003). Comparing RS variables for the UK with those correlates that predict C. imicola abundance in Portugal (Fig. 4b.) illustrates that, at present, establishment of C. imicola in the UK is extremely unlikely in all but a handful of sites, and, in these, only intermediate levels of abundance (which are likely to be of limited epidemiological importance) are predicted. It is noticeable, however, that the south-east of the UK has undergone a significant increase in annual minimum temperatures (Figure 5). This is in common with areas of recent BTV expansion including central and Eastern Europe, some of which areas lack C. imicola as a vector. With or without a further northwards expansion of C. imicola, therefore, the continuation of this trend necessitates a re-assessment of the risk of incursion into and establishment in the UK of BTV via the C. obsoletus and/or C. pulicaris complexes.
Figure 5. Climate change in Europe: Changes in annual minimum temperatures in 0.1C between the 1990’s and 1980’s for each 0.5 square of longitude and latitude, on a sliding scale ranging from a reduction of 2.0C (dark blue) to an increase of 2C (dark red).
Discussion Since the inception of this project, the situation with regard to tracking the vectors of BTV has been complicated by the identification of the Palaearctic C. obsoletus and C. pulicaris species complexes as agents of virus transmission. Prior to the 1998-2005 BTV incursions these Culicoides complexes were thought to be of limited epidemiological importance, however, the recorded absence of C. imicola in certain areas of BTV transmission (e.g. Bulgaria, European Turkey, the Balkans and north-western Greece: see Purse et al., 2005 for review), together with the novel data given in the current report (objectives 1&2), form a strong case for the involvement of the C. obsoletus and C. pulicaris species complexes. in BTV transmission in Europe. The involvement of populations of these complexes in BTV transmission has been brought about by the northerly expansion of C. imicola across most of southern Europe to include much of Italy, Spain and parts of southern France so that the distributions of the two sets of vectors significantly overlap which has facilitated a continuum for virus transmission. This has led to BTV being able to penetrate much further into Europe than ever before (i.e. as far as 44o 30’N). The further northerly expansion in the range of C. imicola as climate-change progresses is probable, though under present climatic conditions its expansion to include the UK within its distribution seems unlikely. However, through the work of this and related projects it is now known that populations of relatively efficient BTV vectors within the C. obsoletus/C. pulicaris species complexes are already present in parts of Europe including the UK. Since these two species complexes occur throughout central and northern Europe, ranging as far north as Scandinavia, this could potentially pose a risk of BTV spreading much further north than hitherto even without the involvement of C. imicola. To date, detailed knowledge regarding almost every aspect of the biology of the C. obsoletus/C. pulicaris groups is lacking. Consequently, the new BBSRC/DEFRA programme already referred to above will undertake a detailed nationwide survey of Culicoides in the UK and will investigate the risk of BTV incursion and establishment, through the use of mathematical modelling, satellite imagery and further vector competence studies, and will also assess control measures. Appendix 1. References.
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