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Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep
Veterinary Microbiology 88 (2002) 115±125
Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep K.R. Bonneaua, C.D. DeMaulaa, B.A. Mullensb, N.J. MacLachlana,* a
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA b Department of Entomology, University of California, Riverside, CA 92521, USA
Received 28 August 2001; received in revised form 15 January 2002; accepted 5 May 2002
Abstract The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111±222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTVas determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RTnested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Bluetongue virus; Duration of viraemia; Culicoides sonorensis; RT-PCR; Cattle-viruses; Sheep-viruses
* Corresponding author. Tel.: 1-530-752-1385; fax: 1-530-754-8124. E-mail address: [email protected] (N.J. MacLachlan).
0378-1135/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 1 0 6 - 2
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1. Introduction Bluetongue virus (BTV) is the prototype member of the genus Orbivirus in the family Reoviridae (Van Regenmortel et al., 2000). BTV is the causative agent of bluetongue, a non-contagious, insect-transmitted disease of sheep and some species of wild ruminants (MacLachlan, 1994). In contrast, BTV infection of cattle is typically asymptomatic (Barratt-Boyes and MacLachlan, 1995). Viraemia is often prolonged in both sheep and cattle because of a novel interaction of BTV with ovine and bovine erythrocytes (Eaton and Crameri, 1989; Brewer and MacLachlan, 1992, 1994; MacLachlan, 1994; Barratt-Boyes and MacLachlan, 1995). Thus, cattle and sheep are considered to be reservoir hosts of BTV from which virus is transmitted to hematophagous insects of the genus Culicoides (Foster et al., 1963; Luedke et al., 1967; MacLachlan, 1994; Barratt-Boyes and MacLachlan, 1995). BTV infection occurs throughout tropical and temperate regions of the world, coincident with the distribution of competent vector insects (Gibbs and Greiner, 1994). Culicoides sonorensis (C. sonorensis; formerly C. variipennis) is the principal vector of BTV in North America (Tabachnick, 1996; Holbrook et al., 2000). Female Culicoides become persistently infected with BTV and transmit the virus to susceptible ruminants after an extrinsic incubation period of approximately 10±14 days (Foster et al., 1963; Luedke et al., 1967). Bluetongue is 1 of only 16 diseases included in List A by the Of®ce International des Epizooties, and so is of major importance to international trade of livestock and germplasm (Alexander et al., 1996). Accurate determination of the risk posed by ruminants previously exposed to BTV clearly is central to the development of rational trade policies pertaining to the virus. Viraemia often is prolonged in BTV-infected ruminants, as determined by conventional virus isolation procedures (Koumbati et al., 1999; Singer et al., 2001). Singer et al. (2001) recently concluded that there is a >99% probability of detectable viraemia ceasing after 9 weeks post-infection (p.i.) in BTV-infected cattle, based on analysis of published data from more than 500 cattle that were naturally or experimentally infected with BTV (Singer et al., 2001). Sheep experimentally infected with BTV also may experience prolonged viraemia that occurs up to 54 days p.i. (Richards et al., 1988; Koumbati et al., 1999). BTV nucleic acids can be detected in ruminant blood for even longer periods (up to 100 and 180 days p.i. of sheep and cattle, respectively) using BTV speci®c reverse transcriptase±polymerase chain reaction (RT-PCR) assays (Katz et al., 1993, 1994), although it is uncertain as to the state in which BTV RNA persists. The RTPCR assay potentially would detect viral nucleic acids contained in any inactivated virions or subviral particles bound to circulating erythrocytes (Brewer and MacLachlan, 1992), however, cattle blood that was positive by RT-PCR assay, but negative by virus isolation was not infectious to inoculated ruminants or to C. sonorensis fed the blood in vitro (MacLachlan et al., 1994). Furthermore, intrathoracic inoculation of vector insects with cattle blood that was positive by RT-PCR assay but negative by virus isolation also failed to establish infection (Tabachnick et al., 1996). Thus, ruminants whose blood contains RT-PCR-detectable BTV nucleic acid, but not infectious virus as determined by virus isolation, likely are unimportant to the epidemiology of BTV infection (MacLachlan et al., 1994; Tabachnick et al., 1996). The duration of BTV viraemia in cattle and sheep that is infectious to C. sonorensis can only be precisely determined by the sequential feeding of susceptible insect vectors
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directly on ruminants that previously were infected with the virus. Such an approach would establish whether the process of C. sonorensis feeding might somehow activate latent virus infection or subviral core particles associated with erythrocytes or other blood cells, thereby resurrecting infectious BTV from ruminant blood that is positive by RT-PCR assay but negative by virus isolation. Thus, the objective of this study was to determine the biological signi®cance of RT-PCR-detectable BTV nucleic acid in ruminant blood by monitoring infection of susceptible C. sonorensis fed directly on cattle and sheep at regular intervals after BTV infection. 2. Materials and methods 2.1. Virus A strain of BTV serotype 17 isolated from the blood of a sheep that died of bluetongue in Tulare County, CA was used to infect C. sonorensis, cattle and sheep. Virus in the blood of the sheep was passaged twice in seronegative cattle and then isolated from cattle blood using primary bovine pulmonary artery endothelial cells, as previously described (DeMaula et al., 2001). The virus then was passaged once in baby hamster kidney (BHK-21) cells to produce a virus stock, and the titer of the stock was determined as tissue culture infectious doses (TCID50) by endpoint titration assay (Reed and Muench, 1938). This strain of BTV is readily isolated in cell culture from the blood of infected ruminants (unpublished observations). 2.2. Insect rearing and feeding A laboratory colony of C. sonorensis Wirth and Jones was established from a southern California ®eld population that is susceptible to BTV infection, as previously described (Mullens et al., 1995; Gerry et al., 2001). Pupae were shipped via overnight mail from the University of California, Riverside, to the University of California, Davis, and adults were reared for the feeding trials. Adult ¯ies were orally infected with BTVat 1±4 days of age by feeding on de®brinated sheep blood spiked with BTV at a titer of 107.1 TCID50 per milliliter. Insects were fed on the spiked blood through a para®lm membrane in a temperature-controlled feeding apparatus (Hunt and McKinnon, 1990). Engorged females were held for a 10-day extrinsic incubation period at 27 8C, and survivors were used to infect susceptible cattle and sheep by feeding for approximately 1 h through a nylon mesh stocking af®xed to a holding cage that was attached to a shaved area on the abdomen of each animal (Bonneau et al., 2001). Female insects that imbibed a second blood meal were pooled and homogenized in grinding buffer (10 mM Tris±HCl, 10 mM NaCl, and 10 mM Na2EDTA, pH 8; ®ve ¯ies per 100 ml) and stored at 7 8C. 2.3. Animals and experimental design Four bulls and four sheep were obtained from northwestern CA, a region free of BTV infection. The animals were approximately 1±2 years of age at the onset of the study.
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Table 1 Number of C. sonorensis in insect pools Species and animal number
Days post-infection 0
7
21
49
Cattle 400 401 402 403e
205a (131)b 157 (45) 29 (5) 90 (12)
11c 72 NAd 69
42 100 123 158
43 56 55 35
Sheep 991 992 993 994e
27 51 151 50
61 13 27 32
15 9 181 17
57 47 32 59
(7) (14) (41) (6)
a
Number of insects taking virus-spiked blood meal. Number of insects taking a second blood meal from each ruminant. c Number of insects taking a blood meal at each feeding. d NA, not available. e Control animals. b
Three bulls (400, 401 and 402) and three sheep (991, 992 and 993) were infected with BTV by the feeding of C. sonorensis that previously had imbibed a blood meal of de®brinated sheep blood spiked with BTV. Surviving female C. sonorensis were fed directly on the skin of the cattle and sheep, as described above (Table 1). Insects fed blood spiked only with tissue culture medium containing lysed BHK-21 cells were fed on a single bull and sheep (403 and 994, respectively) as uninfected controls. Blood was collected from all animals at 0, 1, 3, 5, 7, 9 and 11 days p.i. and thereafter at approximately 2, 3, 7, 12, 16, 17, 20, 24 and 32 weeks p.i. for virus isolation in BHK-21 cells, BTV-speci®c RT-nested PCR assay, and serology. Groups of approximately 1000 uninfected C. sonorensis subsequently were fed on individual cattle and sheep for 1±2 h at 7, 21 and 49 days p.i., sorted on the basis of blood feeding, and held for a 10-day extrinsic incubation period. Survivors within each group were pooled, homogenized in grinding buffer, and stored at 70 8C (Table 1). Virus isolation techniques and RT-PCR assay utilizing BTV-speci®c primers were used to detect the presence of BTV and/or BTV nucleic acid. All cattle and sheep were housed in insectsecure isolation facilities. 2.4. Serology The presence of BTV-speci®c antibodies in the serum of cattle and sheep was determined at 14 days p.i. using a competitive enzyme-linked immunosorbent assay (cELISA; Blueplate Special, DiagXotics, Wilton, CT). This test detects virus-speci®c antibodies in the serum of ruminants that previously were infected with BTV by quantitating the ability of individual sera to displace the binding of a monoclonal antibody speci®c for BTV core protein VP7 (Reddington et al., 1991).
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2.5. Virus isolation Virus isolation was performed on ruminant blood and homogenized insects. Blood samples were collected and processed as previously described (Heidner et al., 1991; Bonneau et al., 2001). Brie¯y, con¯uent monolayers of BHK-21 cells in 25 cm2 ¯asks were inoculated with 1 ml of washed, lysed blood cells and incubated at 37 8C for 10 days. For virus isolations from C. sonorensis, homogenates of pooled insects were sonicated and diluted to approximately 4 ml with minimal essential medium and inoculated onto con¯uent monolayers of BHK-21 cells that were maintained in medium containing antimicrobial agents (2.5 mg/ml amphotericin B, 200 mg/ml penicillin and streptomycin,100 mg/ml neomycin sulfate, and 100 mg/ml gentamicin sulfate) and incubated at 37 8C for 10 days. Cultures that did not exhibit cytopathic effect were passaged a second time. Cytopathic agents isolated from ruminant blood and/or insect homogenates were con®rmed as BTV by immuno¯uorescent staining of infected monolayers of BHK-21 cells grown on chamber slides using a FITC-conjugated monoclonal antibody to BTV core protein VP7 (Whetter et al., 1989). Samples were considered negative after two serial passages without cytopathic effect. Titers of BTV in the blood of bull 401 were determined at 5, 7, 9, 14 and 21 days p.i., and in the blood of sheep 993 at 3, 5, 7, 9 and 21 days p.i., as previously described (Bonneau et al., 2001). Brie¯y, con¯uent monolayers of BHK-21 cells in 24-well plates were inoculated in triplicate with serial 10-fold dilutions of lysed, washed blood cells followed by incubation at 37 8C for 10 days. Titers were calculated as TCID50 per milliliter of blood by the method of Reed and Muench (1938). A sheep (995) that was seronegative for BTV-speci®c antibodies by cELISA was inoculated with blood collected from bulls 401, 402 and 403 at approximately 120 days p.i., and from sheep 991, 992 and 993 at 49 days p.i. Another seronegative sheep (996) was inoculated with homogenized insects that fed on the BTV-inoculated cattle and sheep at 49 days p.i. Volumes of blood and homogenized insects used to inoculate each animal were identical to those used for virus isolation in cell culture. The serological status of these two sheep was determined by cELISA at 14 and 28 days p.i. 2.6. RNA extraction and RT-PCR analysis Total RNA was isolated directly from ruminant blood using RNA STAT-50 LSTM (Tel Test Inc., Friendswood, TX) according to the manufacturer's protocol, and from homogenized insects in grinding buffer as previously described (Wilson and Chase, 1993). Total RNA isolated from blood was screened for the presence of BTV nucleic acids using a previously described RT-nested PCR protocol (Wilson and Chase, 1993), with the modi®cation that a 775 bp portion of the NS3/NS3A gene (genome segment 10) was ampli®ed using BTV-speci®c primers (Bonneau et al., 2001). NS3/NS3A gene cDNA was synthesized and ampli®ed with primers designed to bind to the extreme 50 and 30 ends of genome segment 10 (S10 50 primer, 50 -GTTAAAAAGTGTCGCTGCCATG-30 , sense; S10 30 primer, 50 -GTAAGTGTGTAGTATCGCGCAC-30 , antisense) which then was used as the template for the nested PCR with primers that ampli®ed a 775 bp region (S10 50 nested primer, 50 -ATCCGGGCTGATCCAAAGGTTCG-30 , sense; S10 30 nested primer,
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50 -CTCCCCCGTTATACAGCAGT-30 , antisense). Total RNA isolated from homogenized insects was screened for the presence of BTV nucleic acids using a protocol identical to that used for detection of BTV RNA in ruminant blood, with the exception that a nested PCR reaction was not used. Nested RT-PCR was not used for screening C. sonorensis pools for BTV RNA as BTV replicates to high titers in its insect host (Mellor, 2000). 3. Results 3.1. Virus isolation and serology Three bulls and three sheep were infected with BTV by the feeding of female C. sonorensis that had been infected by ingesting a BTV-spiked blood meal. Although the number of infected insects that survived the extrinsic incubation period and fed on each ruminant varied (Table 1), all six animals seroconverted to BTV by 14 days after exposure to infective insects. Virus was ®rst isolated from bulls 400 and 402 at 7 days p.i., and viraemia persisted in these two bulls until 22 and 21 days p.i., respectively, as determined by virus isolation in BHK-21 cells (Fig. 1). BTV was ®rst detected by virus isolation in the blood of bull 401 at 14 days p.i. and persisted until 49 days p.i. Virus was ®rst detected by virus isolation in the blood of all three sheep (991, 992 and 993) at 5 days p.i., and was not isolated after 11 days p.i. (Fig. 1). BTV was not isolated from the blood of either the control bull (403) or sheep (994), nor did either animal seroconvert to BTV.
Fig. 1. Detection of BTV in the blood of bulls (cattle) and sheep infected with BTV 17 by insect inoculation, as determined by reverse transcriptase-nested PCR analysis (RT-nPCR), and by virus isolation (VI) on BHK-21 cells (VI from cattle or sheep blood). The duration of viraemia infectious to C. sonorensis fed on the bulls and sheep subsequent to BTV inoculation was determined by virus isolation from groups of insects fed at 7, 21, and 49 days p.i. (VI from C. sonorensis). (): only insects fed on bull 401 at 21 days p.i. contained infectious BTV as determined by virus isolation, and not those fed at 7 days p.i. Bull 403 and sheep 994 are uninfected (virus-negative) controls.
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Table 2 Detection of bluetongue virus in C. sonorensis fed on cattle and sheep Insect group
Days post-infection 0a
Cattle 400 401 402 403 Sheep 991 992 993 994
VIb RT-PCRc VI RT-PCR VI RT-PCR VI RT-PCR
VI RT-PCR VI RT-PCR VI RT-PCR VI RT-PCR
7
NAd NA
21
49
a
Insects used to infect cattle and sheep. VI, virus isolation. c RT-PCR, reverse transcriptase±polymerase chain reaction. d NA, not available. b
Groups of insects were fed on the BTV-infected cattle and sheep at 7, 21 and 49 days p.i. (Table 1). BTV was isolated in BHK-21 cells from 10 pools of insects, including the 6 pools of insects that imbibed the BTV-spiked blood meal and were used to infect the cattle and sheep (Table 2). Insects that fed on bull 401 at 21 days p.i. contained BTV as determined by virus isolation, whereas the insects that fed on this same bull at 7 days and 49 days p.i. did not (Table 2 and Fig. 1). Insects that fed on sheep 991 at 7 days p.i. contained infectious BTV, as did insects that fed on sheep 993 at 7 and 21 days p.i. Insects that fed on sheep 991 at 21 and 49 days p.i. did not contain BTV as determined by virus isolation, nor did insects that fed on sheep 993 at 49 days p.i. BTV was not isolated from any insects that fed on bulls 400 and 402, or sheep 992, despite the fact that all of these animals became viremic as determined by virus isolation (Fig. 1). None of the insects that fed on the uninfected control animals contained BTV as determined by virus isolation. Titers of BTV were quantitated in the blood of bull 401 and sheep 993. The insects that fed on bull 401 at 7 days p.i. imbibed blood from which virus was not isolated (<100.5 TCID50 per milliliter (the threshold of the titration assay)) and did not become infected with BTV, whereas those that fed from the same animal at 21 days p.i. became infected after imbibing blood that contained BTV at a concentration of 101.4 TCID50 per milliliter (Table 2). The groups of insects that fed on sheep 993 at 7 and 21 days p.i. imbibed blood,
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respectively, containing 102.4 and <100.5 TCID50 per milliliter, and both groups of insects became infected with BTV (Table 2). The two sheep (995 and 996) that, respectively, were inoculated with pooled ruminant blood and homogenized insects remained seronegative to BTV, con®rming that infectious virus was not present in the blood of either the three BTV-infected cattle at approximately 120 days p.i. or the three BTV-infected sheep at 49 days p.i. when all blood samples were negative by virus isolation, but clearly positive by RT-nested PCR assay. Insects that fed on these same six animals at 49 days p.i. also did not contain BTV as determined by virus isolation in cell culture, RT-PCR, and direct inoculation of a susceptible sheep (Table 2). 3.2. Detection of BTV nucleic acid by RT-PCR assay BTV nucleic acids were ®rst detected by RT-nested PCR in the blood of bulls 400, 401 and 402 at 5, 3 and 7 days p.i., respectively, and were continuously detected until 144±167 days p.i. (Fig. 1). BTV nucleic acids were ®rst detected in the blood of infected sheep 991, 992 and 993 by RT-nested PCR at 3 days p.i., and were continuously detected until 111±222 days p.i. (Fig. 1). BTV nucleic acids were not detected at any time in the blood of either the control bull (403) or sheep (994). BTV nucleic acids were detected by RT-PCR only in insects pools from which BTV also was isolated in BHK-21 cells. All six groups of insects that were infected with BTV by imbibing a virus-spiked blood meal and subsequently used to infect the three bulls and three sheep by oral feeding were positive by RT-PCR, whereas the control insects that ingested blood spiked only with tissue culture medium containing lysed BHK-21 cells were not. Of the insects that were fed on the infected cattle at 7, 21 and 49 days p.i., BTV nucleic acids were only detected by RT-PCR in those insects that fed on bull 401 at 21 days p.i. and this same group of insects was also positive for BTV by virus isolation (Table 2). Of the insects that were fed on the infected sheep at 7, 21 and 49 days p.i., BTV nucleic acids were only detected by RT-PCR in the insects that fed on sheep 991 at 7 days p.i. and those that fed on sheep 993 at 7 and 21 days p.i. These same groups of insects also were positive for BTV by virus isolation (Table 2). The insects that fed on bulls 400 and 402, as well as those that fed on sheep 992, all were negative for the presence of BTV nucleic acids by RT-PCR, as were all pools of insects that fed on the control bull and sheep (bull 403 and sheep 994, Table 2). 4. Discussion More precise determination of the duration of viraemia in BTV-infected ruminants that is infectious to vector insects is prerequisite to the design of logical trade policies pertaining to the virus (Alexander et al., 1996; Singer et al., 2001). Previous studies have shown that cattle blood that contained BTV nucleic acid but not infectious virus is not infectious to either ruminants or the insect vector, even by intrathoracic inoculation of C. sonorensis which bypasses the insect's midgut barrier (MacLachlan et al., 1994; Tabachnick et al., 1996; Fu et al., 1999; Mellor, 2000). In contrast, cattle blood that is positive by both virus isolation and RT-PCR techniques is infectious to C. sonorensis, following either oral feeding
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in vitro or intrathoracic inoculation (MacLachlan et al., 1994; Tabachnick et al., 1996). Thus, cattle whose blood contains BTV nucleic acid as determined by RT-PCR analysis, but not infectious virus as determined by virus isolation techniques, are presumably unimportant to the epidemiology of BTV infection. In the current study, we have further investigated the duration of BTV viraemia in cattle and sheep that is infectious to the insect vector by the sequential feeding of C. sonorensis directly on infected ruminants. The virus isolation procedure using BHK-21 cells more reliably identi®ed the presence of infectious BTV in the blood of infected sheep and cattle than did the feeding of vector insects. The insects used in this study were derived directly from a competent ®eld population (Gerry et al., 2001), and had not been selected for high competence through development of susceptible single-family lines that were used in some earlier laboratory studies (Mullens et al., 1995). All six pools of insects that imbibed the high-titered, BTVspiked blood were infected with BTV whereas only 4 of the 18 pools of insects that fed on the 6 BTV-infected cattle and sheep at 7, 21 and 49 days p.i. contained BTV as determined by virus isolation and RT-PCR assays. Furthermore, although the insects contained in 10 of the 18 pools had clearly fed on ruminants that were viraemic, as determined by virus isolation from the blood of these animals (Fig. 1), only 3 of these 10 insect pools contained BTV (Table 2). Titers of BTV in both cattle and sheep blood (<100.5 to 102.4 TCID50 per milliliter) were signi®cantly lower than that of the spiked blood (107.1 TCID50 per milliliter). Thus, the low titer of BTV in the blood of infected cattle and sheep, the small blood meal taken by each insect, and the use of an insect population not arti®cially selected for high competence all likely contribute to the sporadic infection of groups of C. sonorensis that fed on these ruminants. Although relatively inef®cient, the infection of these laboratory-reared vector insects by feeding on infected ruminants is similar to that which occurs during natural transmission of BTV where much larger populations of vector insects are involved (Gerry et al., 2001). Not only did the virus isolation procedure we used in this study more reliably detect the presence of BTV in the blood of ruminants than did the feeding of vector insects but, with the notable exception of sheep 993, insects that fed on ruminants whose blood was positive by RT-nested PCR assay, but not by virus isolation did not become infected with BTV. However, ruminant blood that is negative by virus isolation yet positive for BTV RNA by RT-nested PCR can occasionally be infective to C. sonorensis, as an insect(s) became infected after feeding on sheep 993 that was positive for BTV RNA by RT-nested PCR, but negative by virus isolation (threshold <100.5 TCID50 per milliliter) at 21 days p.i. This occurrence likely re¯ects a sampling event when titers of BTV in the blood of infected ruminants are very low (Barratt-Boyes and MacLachlan, 1995), although the likelihood of such an event occurring in the ®eld (natural) situation is presumably higher because of the greater numbers of competent vector insects that feed on ruminants in this setting (Gerry et al., 2001). In summary, results of the present study further con®rm that infectious viremia is prolonged but transient in BTV-infected cattle and sheep. Furthermore, ruminants whose blood is con®rmed to be free of BTV by both virus isolation and RT-nested PCR assays are clearly not infective to vector insects, thus the RT-nested PCR procedure is an extremely sensitive, stringent and very conservative assay that reliably can be used to screen ruminants prior to exportation to BTV-free regions.
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Acknowledgements The authors thank Drs. Udeni Balasuriya, Brian Moore and Jodi Hedges for technical support and manuscript review, Bridget McLaughlin, J.B. Topol and Andrea Navarette for laboratory assistance, Heike Wagner for veterinary assistance, and Dr. Alec Gerry and Robert Velten for propagation and maintenance of insects. These studies were supported by USDA-NRI Competitive Grant #99-35204-7863, funds provided by the Center for Food Animal Health, and the USDA under the Animal Health Act, 1977, Public Law 95±113.
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Koumbati, M., Mangana, O., Nomikou, K., Mellor, P.S., Papadopoulos, O., 1999. Duration of bluetongue viraemia and serological responses in experimentally infected European breeds of sheep and goats. Vet. Microbiol. 64, 277±285. Luedke, A.J., Jones, R.H., Jochim, M.M., 1967. Transmission of bluetongue between sheep and cattle by Culicoides variipennis. Am. J. Vet. Res. 28, 457±460. MacLachlan, N.J., 1994. The pathogenesis and immunology of bluetongue virus infection of ruminants. Comp. Immunol. Microbiol. Infect. Dis. 17, 197±206. MacLachlan, N.J., Nunamaker, R.A., Katz, J.B., Sawyer, M.M., Akita, G.Y., Osburn, B.I., Tabachnick, W.J., 1994. Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis. Arch. Virol. 136, 1±8. Mellor, P.S., 2000. Replication of arboviruses in insect vectors. J. Comp. Pathol. 123, 231±247. Mullens, B.A., Tabachnick, W.J., Holbrook, F.R., Thompson, L.H., 1995. Effects of temperature on virogenesis of bluetongue virus serotype 11 in Culicoides vanipennis sonorensis. Med. Vet. Entomol. 9, 71±76. Reddington, J.J., Reddington, G., MacLachlan, N.J., 1991. A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus. J. Vet. Diagn. Invest. 3, 144±147. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493±497. Richards, R.G., MacLachlan, N.J., Heidner, H.W., Fuller, F.J., 1988. Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10. Vet. Microbiol. 18, 233±242. Singer, R.S., MacLachlan, N.J., Carpenter, T.E., 2001. Maximal predicted duration of viremia in bluetongue virus-infected cattle. J. Vet. Diag. Invest. 13, 43±49. Tabachnick, W.J., 1996. Culicoides variipennis and bluetongue-virus epidemiology in the United States. Trans. R. Soc. Trop. Med. Hyg. 41, 23±43. Tabachnick, W.J., MacLachlan, N.J., Thompson, L.H., Hunt, G.J., Patton, J.F., 1996. Susceptibility of Culicoides variipennis sonorensis to infection by polymerase chain reaction-detectable bluetongue virus in cattle blood. Am. J. Trop. Med. Hyg. 54, 481±485. Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., McGeoch, D.J., Maniloff, J., Mayo, M.A., Pringle, C.R., Wickner, R.B. (Eds.), Seventh ICTV Report. Academic Press, New York, San Diego, pp. 408±421. Whetter, L.E., MacLachlan, N.J., Gebliard, D.H., Heidner, H.W., Moore, P.F., 1989. Bluetongue virus infection of bovine monocytes. J. Gen. Virol. 70, 1663±1676. Wilson, W.C., Chase, C.C., 1993. Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis. J. Virol. Meth. 45, 39±47.
Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep K.R. Bonneaua, C.D. DeMaulaa, B.A. Mullensb, N.J. MacLachlana,* a
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA b Department of Entomology, University of California, Riverside, CA 92521, USA
Received 28 August 2001; received in revised form 15 January 2002; accepted 5 May 2002
Abstract The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111±222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTVas determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RTnested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Bluetongue virus; Duration of viraemia; Culicoides sonorensis; RT-PCR; Cattle-viruses; Sheep-viruses
* Corresponding author. Tel.: 1-530-752-1385; fax: 1-530-754-8124. E-mail address: [email protected] (N.J. MacLachlan).
0378-1135/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 1 0 6 - 2
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1. Introduction Bluetongue virus (BTV) is the prototype member of the genus Orbivirus in the family Reoviridae (Van Regenmortel et al., 2000). BTV is the causative agent of bluetongue, a non-contagious, insect-transmitted disease of sheep and some species of wild ruminants (MacLachlan, 1994). In contrast, BTV infection of cattle is typically asymptomatic (Barratt-Boyes and MacLachlan, 1995). Viraemia is often prolonged in both sheep and cattle because of a novel interaction of BTV with ovine and bovine erythrocytes (Eaton and Crameri, 1989; Brewer and MacLachlan, 1992, 1994; MacLachlan, 1994; Barratt-Boyes and MacLachlan, 1995). Thus, cattle and sheep are considered to be reservoir hosts of BTV from which virus is transmitted to hematophagous insects of the genus Culicoides (Foster et al., 1963; Luedke et al., 1967; MacLachlan, 1994; Barratt-Boyes and MacLachlan, 1995). BTV infection occurs throughout tropical and temperate regions of the world, coincident with the distribution of competent vector insects (Gibbs and Greiner, 1994). Culicoides sonorensis (C. sonorensis; formerly C. variipennis) is the principal vector of BTV in North America (Tabachnick, 1996; Holbrook et al., 2000). Female Culicoides become persistently infected with BTV and transmit the virus to susceptible ruminants after an extrinsic incubation period of approximately 10±14 days (Foster et al., 1963; Luedke et al., 1967). Bluetongue is 1 of only 16 diseases included in List A by the Of®ce International des Epizooties, and so is of major importance to international trade of livestock and germplasm (Alexander et al., 1996). Accurate determination of the risk posed by ruminants previously exposed to BTV clearly is central to the development of rational trade policies pertaining to the virus. Viraemia often is prolonged in BTV-infected ruminants, as determined by conventional virus isolation procedures (Koumbati et al., 1999; Singer et al., 2001). Singer et al. (2001) recently concluded that there is a >99% probability of detectable viraemia ceasing after 9 weeks post-infection (p.i.) in BTV-infected cattle, based on analysis of published data from more than 500 cattle that were naturally or experimentally infected with BTV (Singer et al., 2001). Sheep experimentally infected with BTV also may experience prolonged viraemia that occurs up to 54 days p.i. (Richards et al., 1988; Koumbati et al., 1999). BTV nucleic acids can be detected in ruminant blood for even longer periods (up to 100 and 180 days p.i. of sheep and cattle, respectively) using BTV speci®c reverse transcriptase±polymerase chain reaction (RT-PCR) assays (Katz et al., 1993, 1994), although it is uncertain as to the state in which BTV RNA persists. The RTPCR assay potentially would detect viral nucleic acids contained in any inactivated virions or subviral particles bound to circulating erythrocytes (Brewer and MacLachlan, 1992), however, cattle blood that was positive by RT-PCR assay, but negative by virus isolation was not infectious to inoculated ruminants or to C. sonorensis fed the blood in vitro (MacLachlan et al., 1994). Furthermore, intrathoracic inoculation of vector insects with cattle blood that was positive by RT-PCR assay but negative by virus isolation also failed to establish infection (Tabachnick et al., 1996). Thus, ruminants whose blood contains RT-PCR-detectable BTV nucleic acid, but not infectious virus as determined by virus isolation, likely are unimportant to the epidemiology of BTV infection (MacLachlan et al., 1994; Tabachnick et al., 1996). The duration of BTV viraemia in cattle and sheep that is infectious to C. sonorensis can only be precisely determined by the sequential feeding of susceptible insect vectors
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directly on ruminants that previously were infected with the virus. Such an approach would establish whether the process of C. sonorensis feeding might somehow activate latent virus infection or subviral core particles associated with erythrocytes or other blood cells, thereby resurrecting infectious BTV from ruminant blood that is positive by RT-PCR assay but negative by virus isolation. Thus, the objective of this study was to determine the biological signi®cance of RT-PCR-detectable BTV nucleic acid in ruminant blood by monitoring infection of susceptible C. sonorensis fed directly on cattle and sheep at regular intervals after BTV infection. 2. Materials and methods 2.1. Virus A strain of BTV serotype 17 isolated from the blood of a sheep that died of bluetongue in Tulare County, CA was used to infect C. sonorensis, cattle and sheep. Virus in the blood of the sheep was passaged twice in seronegative cattle and then isolated from cattle blood using primary bovine pulmonary artery endothelial cells, as previously described (DeMaula et al., 2001). The virus then was passaged once in baby hamster kidney (BHK-21) cells to produce a virus stock, and the titer of the stock was determined as tissue culture infectious doses (TCID50) by endpoint titration assay (Reed and Muench, 1938). This strain of BTV is readily isolated in cell culture from the blood of infected ruminants (unpublished observations). 2.2. Insect rearing and feeding A laboratory colony of C. sonorensis Wirth and Jones was established from a southern California ®eld population that is susceptible to BTV infection, as previously described (Mullens et al., 1995; Gerry et al., 2001). Pupae were shipped via overnight mail from the University of California, Riverside, to the University of California, Davis, and adults were reared for the feeding trials. Adult ¯ies were orally infected with BTVat 1±4 days of age by feeding on de®brinated sheep blood spiked with BTV at a titer of 107.1 TCID50 per milliliter. Insects were fed on the spiked blood through a para®lm membrane in a temperature-controlled feeding apparatus (Hunt and McKinnon, 1990). Engorged females were held for a 10-day extrinsic incubation period at 27 8C, and survivors were used to infect susceptible cattle and sheep by feeding for approximately 1 h through a nylon mesh stocking af®xed to a holding cage that was attached to a shaved area on the abdomen of each animal (Bonneau et al., 2001). Female insects that imbibed a second blood meal were pooled and homogenized in grinding buffer (10 mM Tris±HCl, 10 mM NaCl, and 10 mM Na2EDTA, pH 8; ®ve ¯ies per 100 ml) and stored at 7 8C. 2.3. Animals and experimental design Four bulls and four sheep were obtained from northwestern CA, a region free of BTV infection. The animals were approximately 1±2 years of age at the onset of the study.
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Table 1 Number of C. sonorensis in insect pools Species and animal number
Days post-infection 0
7
21
49
Cattle 400 401 402 403e
205a (131)b 157 (45) 29 (5) 90 (12)
11c 72 NAd 69
42 100 123 158
43 56 55 35
Sheep 991 992 993 994e
27 51 151 50
61 13 27 32
15 9 181 17
57 47 32 59
(7) (14) (41) (6)
a
Number of insects taking virus-spiked blood meal. Number of insects taking a second blood meal from each ruminant. c Number of insects taking a blood meal at each feeding. d NA, not available. e Control animals. b
Three bulls (400, 401 and 402) and three sheep (991, 992 and 993) were infected with BTV by the feeding of C. sonorensis that previously had imbibed a blood meal of de®brinated sheep blood spiked with BTV. Surviving female C. sonorensis were fed directly on the skin of the cattle and sheep, as described above (Table 1). Insects fed blood spiked only with tissue culture medium containing lysed BHK-21 cells were fed on a single bull and sheep (403 and 994, respectively) as uninfected controls. Blood was collected from all animals at 0, 1, 3, 5, 7, 9 and 11 days p.i. and thereafter at approximately 2, 3, 7, 12, 16, 17, 20, 24 and 32 weeks p.i. for virus isolation in BHK-21 cells, BTV-speci®c RT-nested PCR assay, and serology. Groups of approximately 1000 uninfected C. sonorensis subsequently were fed on individual cattle and sheep for 1±2 h at 7, 21 and 49 days p.i., sorted on the basis of blood feeding, and held for a 10-day extrinsic incubation period. Survivors within each group were pooled, homogenized in grinding buffer, and stored at 70 8C (Table 1). Virus isolation techniques and RT-PCR assay utilizing BTV-speci®c primers were used to detect the presence of BTV and/or BTV nucleic acid. All cattle and sheep were housed in insectsecure isolation facilities. 2.4. Serology The presence of BTV-speci®c antibodies in the serum of cattle and sheep was determined at 14 days p.i. using a competitive enzyme-linked immunosorbent assay (cELISA; Blueplate Special, DiagXotics, Wilton, CT). This test detects virus-speci®c antibodies in the serum of ruminants that previously were infected with BTV by quantitating the ability of individual sera to displace the binding of a monoclonal antibody speci®c for BTV core protein VP7 (Reddington et al., 1991).
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2.5. Virus isolation Virus isolation was performed on ruminant blood and homogenized insects. Blood samples were collected and processed as previously described (Heidner et al., 1991; Bonneau et al., 2001). Brie¯y, con¯uent monolayers of BHK-21 cells in 25 cm2 ¯asks were inoculated with 1 ml of washed, lysed blood cells and incubated at 37 8C for 10 days. For virus isolations from C. sonorensis, homogenates of pooled insects were sonicated and diluted to approximately 4 ml with minimal essential medium and inoculated onto con¯uent monolayers of BHK-21 cells that were maintained in medium containing antimicrobial agents (2.5 mg/ml amphotericin B, 200 mg/ml penicillin and streptomycin,100 mg/ml neomycin sulfate, and 100 mg/ml gentamicin sulfate) and incubated at 37 8C for 10 days. Cultures that did not exhibit cytopathic effect were passaged a second time. Cytopathic agents isolated from ruminant blood and/or insect homogenates were con®rmed as BTV by immuno¯uorescent staining of infected monolayers of BHK-21 cells grown on chamber slides using a FITC-conjugated monoclonal antibody to BTV core protein VP7 (Whetter et al., 1989). Samples were considered negative after two serial passages without cytopathic effect. Titers of BTV in the blood of bull 401 were determined at 5, 7, 9, 14 and 21 days p.i., and in the blood of sheep 993 at 3, 5, 7, 9 and 21 days p.i., as previously described (Bonneau et al., 2001). Brie¯y, con¯uent monolayers of BHK-21 cells in 24-well plates were inoculated in triplicate with serial 10-fold dilutions of lysed, washed blood cells followed by incubation at 37 8C for 10 days. Titers were calculated as TCID50 per milliliter of blood by the method of Reed and Muench (1938). A sheep (995) that was seronegative for BTV-speci®c antibodies by cELISA was inoculated with blood collected from bulls 401, 402 and 403 at approximately 120 days p.i., and from sheep 991, 992 and 993 at 49 days p.i. Another seronegative sheep (996) was inoculated with homogenized insects that fed on the BTV-inoculated cattle and sheep at 49 days p.i. Volumes of blood and homogenized insects used to inoculate each animal were identical to those used for virus isolation in cell culture. The serological status of these two sheep was determined by cELISA at 14 and 28 days p.i. 2.6. RNA extraction and RT-PCR analysis Total RNA was isolated directly from ruminant blood using RNA STAT-50 LSTM (Tel Test Inc., Friendswood, TX) according to the manufacturer's protocol, and from homogenized insects in grinding buffer as previously described (Wilson and Chase, 1993). Total RNA isolated from blood was screened for the presence of BTV nucleic acids using a previously described RT-nested PCR protocol (Wilson and Chase, 1993), with the modi®cation that a 775 bp portion of the NS3/NS3A gene (genome segment 10) was ampli®ed using BTV-speci®c primers (Bonneau et al., 2001). NS3/NS3A gene cDNA was synthesized and ampli®ed with primers designed to bind to the extreme 50 and 30 ends of genome segment 10 (S10 50 primer, 50 -GTTAAAAAGTGTCGCTGCCATG-30 , sense; S10 30 primer, 50 -GTAAGTGTGTAGTATCGCGCAC-30 , antisense) which then was used as the template for the nested PCR with primers that ampli®ed a 775 bp region (S10 50 nested primer, 50 -ATCCGGGCTGATCCAAAGGTTCG-30 , sense; S10 30 nested primer,
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50 -CTCCCCCGTTATACAGCAGT-30 , antisense). Total RNA isolated from homogenized insects was screened for the presence of BTV nucleic acids using a protocol identical to that used for detection of BTV RNA in ruminant blood, with the exception that a nested PCR reaction was not used. Nested RT-PCR was not used for screening C. sonorensis pools for BTV RNA as BTV replicates to high titers in its insect host (Mellor, 2000). 3. Results 3.1. Virus isolation and serology Three bulls and three sheep were infected with BTV by the feeding of female C. sonorensis that had been infected by ingesting a BTV-spiked blood meal. Although the number of infected insects that survived the extrinsic incubation period and fed on each ruminant varied (Table 1), all six animals seroconverted to BTV by 14 days after exposure to infective insects. Virus was ®rst isolated from bulls 400 and 402 at 7 days p.i., and viraemia persisted in these two bulls until 22 and 21 days p.i., respectively, as determined by virus isolation in BHK-21 cells (Fig. 1). BTV was ®rst detected by virus isolation in the blood of bull 401 at 14 days p.i. and persisted until 49 days p.i. Virus was ®rst detected by virus isolation in the blood of all three sheep (991, 992 and 993) at 5 days p.i., and was not isolated after 11 days p.i. (Fig. 1). BTV was not isolated from the blood of either the control bull (403) or sheep (994), nor did either animal seroconvert to BTV.
Fig. 1. Detection of BTV in the blood of bulls (cattle) and sheep infected with BTV 17 by insect inoculation, as determined by reverse transcriptase-nested PCR analysis (RT-nPCR), and by virus isolation (VI) on BHK-21 cells (VI from cattle or sheep blood). The duration of viraemia infectious to C. sonorensis fed on the bulls and sheep subsequent to BTV inoculation was determined by virus isolation from groups of insects fed at 7, 21, and 49 days p.i. (VI from C. sonorensis). (): only insects fed on bull 401 at 21 days p.i. contained infectious BTV as determined by virus isolation, and not those fed at 7 days p.i. Bull 403 and sheep 994 are uninfected (virus-negative) controls.
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Table 2 Detection of bluetongue virus in C. sonorensis fed on cattle and sheep Insect group
Days post-infection 0a
Cattle 400 401 402 403 Sheep 991 992 993 994
VIb RT-PCRc VI RT-PCR VI RT-PCR VI RT-PCR
VI RT-PCR VI RT-PCR VI RT-PCR VI RT-PCR
7
NAd NA
21
49
a
Insects used to infect cattle and sheep. VI, virus isolation. c RT-PCR, reverse transcriptase±polymerase chain reaction. d NA, not available. b
Groups of insects were fed on the BTV-infected cattle and sheep at 7, 21 and 49 days p.i. (Table 1). BTV was isolated in BHK-21 cells from 10 pools of insects, including the 6 pools of insects that imbibed the BTV-spiked blood meal and were used to infect the cattle and sheep (Table 2). Insects that fed on bull 401 at 21 days p.i. contained BTV as determined by virus isolation, whereas the insects that fed on this same bull at 7 days and 49 days p.i. did not (Table 2 and Fig. 1). Insects that fed on sheep 991 at 7 days p.i. contained infectious BTV, as did insects that fed on sheep 993 at 7 and 21 days p.i. Insects that fed on sheep 991 at 21 and 49 days p.i. did not contain BTV as determined by virus isolation, nor did insects that fed on sheep 993 at 49 days p.i. BTV was not isolated from any insects that fed on bulls 400 and 402, or sheep 992, despite the fact that all of these animals became viremic as determined by virus isolation (Fig. 1). None of the insects that fed on the uninfected control animals contained BTV as determined by virus isolation. Titers of BTV were quantitated in the blood of bull 401 and sheep 993. The insects that fed on bull 401 at 7 days p.i. imbibed blood from which virus was not isolated (<100.5 TCID50 per milliliter (the threshold of the titration assay)) and did not become infected with BTV, whereas those that fed from the same animal at 21 days p.i. became infected after imbibing blood that contained BTV at a concentration of 101.4 TCID50 per milliliter (Table 2). The groups of insects that fed on sheep 993 at 7 and 21 days p.i. imbibed blood,
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respectively, containing 102.4 and <100.5 TCID50 per milliliter, and both groups of insects became infected with BTV (Table 2). The two sheep (995 and 996) that, respectively, were inoculated with pooled ruminant blood and homogenized insects remained seronegative to BTV, con®rming that infectious virus was not present in the blood of either the three BTV-infected cattle at approximately 120 days p.i. or the three BTV-infected sheep at 49 days p.i. when all blood samples were negative by virus isolation, but clearly positive by RT-nested PCR assay. Insects that fed on these same six animals at 49 days p.i. also did not contain BTV as determined by virus isolation in cell culture, RT-PCR, and direct inoculation of a susceptible sheep (Table 2). 3.2. Detection of BTV nucleic acid by RT-PCR assay BTV nucleic acids were ®rst detected by RT-nested PCR in the blood of bulls 400, 401 and 402 at 5, 3 and 7 days p.i., respectively, and were continuously detected until 144±167 days p.i. (Fig. 1). BTV nucleic acids were ®rst detected in the blood of infected sheep 991, 992 and 993 by RT-nested PCR at 3 days p.i., and were continuously detected until 111±222 days p.i. (Fig. 1). BTV nucleic acids were not detected at any time in the blood of either the control bull (403) or sheep (994). BTV nucleic acids were detected by RT-PCR only in insects pools from which BTV also was isolated in BHK-21 cells. All six groups of insects that were infected with BTV by imbibing a virus-spiked blood meal and subsequently used to infect the three bulls and three sheep by oral feeding were positive by RT-PCR, whereas the control insects that ingested blood spiked only with tissue culture medium containing lysed BHK-21 cells were not. Of the insects that were fed on the infected cattle at 7, 21 and 49 days p.i., BTV nucleic acids were only detected by RT-PCR in those insects that fed on bull 401 at 21 days p.i. and this same group of insects was also positive for BTV by virus isolation (Table 2). Of the insects that were fed on the infected sheep at 7, 21 and 49 days p.i., BTV nucleic acids were only detected by RT-PCR in the insects that fed on sheep 991 at 7 days p.i. and those that fed on sheep 993 at 7 and 21 days p.i. These same groups of insects also were positive for BTV by virus isolation (Table 2). The insects that fed on bulls 400 and 402, as well as those that fed on sheep 992, all were negative for the presence of BTV nucleic acids by RT-PCR, as were all pools of insects that fed on the control bull and sheep (bull 403 and sheep 994, Table 2). 4. Discussion More precise determination of the duration of viraemia in BTV-infected ruminants that is infectious to vector insects is prerequisite to the design of logical trade policies pertaining to the virus (Alexander et al., 1996; Singer et al., 2001). Previous studies have shown that cattle blood that contained BTV nucleic acid but not infectious virus is not infectious to either ruminants or the insect vector, even by intrathoracic inoculation of C. sonorensis which bypasses the insect's midgut barrier (MacLachlan et al., 1994; Tabachnick et al., 1996; Fu et al., 1999; Mellor, 2000). In contrast, cattle blood that is positive by both virus isolation and RT-PCR techniques is infectious to C. sonorensis, following either oral feeding
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in vitro or intrathoracic inoculation (MacLachlan et al., 1994; Tabachnick et al., 1996). Thus, cattle whose blood contains BTV nucleic acid as determined by RT-PCR analysis, but not infectious virus as determined by virus isolation techniques, are presumably unimportant to the epidemiology of BTV infection. In the current study, we have further investigated the duration of BTV viraemia in cattle and sheep that is infectious to the insect vector by the sequential feeding of C. sonorensis directly on infected ruminants. The virus isolation procedure using BHK-21 cells more reliably identi®ed the presence of infectious BTV in the blood of infected sheep and cattle than did the feeding of vector insects. The insects used in this study were derived directly from a competent ®eld population (Gerry et al., 2001), and had not been selected for high competence through development of susceptible single-family lines that were used in some earlier laboratory studies (Mullens et al., 1995). All six pools of insects that imbibed the high-titered, BTVspiked blood were infected with BTV whereas only 4 of the 18 pools of insects that fed on the 6 BTV-infected cattle and sheep at 7, 21 and 49 days p.i. contained BTV as determined by virus isolation and RT-PCR assays. Furthermore, although the insects contained in 10 of the 18 pools had clearly fed on ruminants that were viraemic, as determined by virus isolation from the blood of these animals (Fig. 1), only 3 of these 10 insect pools contained BTV (Table 2). Titers of BTV in both cattle and sheep blood (<100.5 to 102.4 TCID50 per milliliter) were signi®cantly lower than that of the spiked blood (107.1 TCID50 per milliliter). Thus, the low titer of BTV in the blood of infected cattle and sheep, the small blood meal taken by each insect, and the use of an insect population not arti®cially selected for high competence all likely contribute to the sporadic infection of groups of C. sonorensis that fed on these ruminants. Although relatively inef®cient, the infection of these laboratory-reared vector insects by feeding on infected ruminants is similar to that which occurs during natural transmission of BTV where much larger populations of vector insects are involved (Gerry et al., 2001). Not only did the virus isolation procedure we used in this study more reliably detect the presence of BTV in the blood of ruminants than did the feeding of vector insects but, with the notable exception of sheep 993, insects that fed on ruminants whose blood was positive by RT-nested PCR assay, but not by virus isolation did not become infected with BTV. However, ruminant blood that is negative by virus isolation yet positive for BTV RNA by RT-nested PCR can occasionally be infective to C. sonorensis, as an insect(s) became infected after feeding on sheep 993 that was positive for BTV RNA by RT-nested PCR, but negative by virus isolation (threshold <100.5 TCID50 per milliliter) at 21 days p.i. This occurrence likely re¯ects a sampling event when titers of BTV in the blood of infected ruminants are very low (Barratt-Boyes and MacLachlan, 1995), although the likelihood of such an event occurring in the ®eld (natural) situation is presumably higher because of the greater numbers of competent vector insects that feed on ruminants in this setting (Gerry et al., 2001). In summary, results of the present study further con®rm that infectious viremia is prolonged but transient in BTV-infected cattle and sheep. Furthermore, ruminants whose blood is con®rmed to be free of BTV by both virus isolation and RT-nested PCR assays are clearly not infective to vector insects, thus the RT-nested PCR procedure is an extremely sensitive, stringent and very conservative assay that reliably can be used to screen ruminants prior to exportation to BTV-free regions.
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Acknowledgements The authors thank Drs. Udeni Balasuriya, Brian Moore and Jodi Hedges for technical support and manuscript review, Bridget McLaughlin, J.B. Topol and Andrea Navarette for laboratory assistance, Heike Wagner for veterinary assistance, and Dr. Alec Gerry and Robert Velten for propagation and maintenance of insects. These studies were supported by USDA-NRI Competitive Grant #99-35204-7863, funds provided by the Center for Food Animal Health, and the USDA under the Animal Health Act, 1977, Public Law 95±113.
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