Seroepidemiologic study of bluetongue virus serotype 2 in Alabama

Preventive Veterinary Medicine, 7 (1989) 113-119 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

113

Seroepidemiologic Study of Bluetongue Virus Serotype 2 in Alabama J.C. WRIGHT 1, L.H. LAUERMAN :~,K.E. NUSBAUM l and G.R. MULLEN 2

Departments of 1Pathobiologyand 2Entomology, Auburn University, AL 36849-5519 (U.S.A.) :~C.S.Roberts Veterinary Diagnostic Laboratory, Department of Agriculture and Industries, Auburn, AL 36831-2209 (U.S.A.) (Accepted for publication 12 October 1988)

ABSTRACT Wright, J.C., Lauerman, L.H., Nusbaum, K.E. and Mullen, G.R., 1989. Seroepidemiologic study of bluetongue virus serotype 2 in Alabama. Prey. Vet. Med., 7: 113-119. Sera from 1341 Alabama cattle representing 65 of 67 counties (97%) were tested for antibodies to bluetongue virus (BTV) using the agar gel precipitin (AGP) test. Those positive by AGP were tested of type-specific antibodies to BTV serotype 2 using the serum neutralization (SN) test. Four hundred and forty-six of the 1341 samples (33%) were positive by AGP. Eighty-four of these 446 samples (19%) were positive for antibodies to BTV serotype 2 (BTV-2). When these sera were tested using the SN test, 28 of the 84 (33%) had titers that were 4-fold greater to BTV-2 than to the other four serotypes of BTV present in the U.S.A. Sera positive for antibodies to BTV2 were from 19 of Alabama's 67 counties (28%).

INTRODUCTION

Bluetongue is a disease of ruminants caused by bluetongue virus (BTV), an orbivirus transmitted mainly by biting midges of the genus Culicoides (du Toit, 1944). Sheep, goats and cattle are primarily affected, but wild ruminants are also susceptible (Erasmus, 1975; House et al., 1982 ). The disease may be either sub-clinical or result in clinical signs of fever, oral lesions, excessive salivation, lameness, abortion, infertility and congenital deformities (Browne, 1971; Erasmus, 1975; Hourrigan and Klingsporn, 1975; Metcalf and Luedke, 1980). Economic losses result from death, infertility and reduced weight gain (Hourrigan and Klingsporn, 1975). Cattle which are disease-free, but serologically positive are an economic burden to cattle exporters because of the restrictions placed on the export of BTV-positive animals (Osburn, 1983). Before 1982, only four serotypes of BTV (10, 11, 13 and 17) were documented in the U.S.A. (Barber, 1979). BTV serotype 2 (BTV-2) was isolated from blood samples collected in September 1982 from asymptomatic sentinel 0167-5877/89/$03.50

© 1989 Elsevier Science Publishers B.V.

114

cattle in Florida (Gibbs et al., 1983). Recently, BTV-2 was isolated from cattle with oral lesions and from an aborted fetus in central and south-eastern Alabama, respectively (Lauerman et al., 1986). The present investigation evaluated the extent of BTV-2 antibodies in cattle in Alabama. M A T E R I A L S AND M E T H O D S

Collection of samples Alabama was divided into five physiographic regions for this survey. Three of these regions (plateau, piedmont, coastal plain) have distinct soil types and topography. Representative sera from each region of Alabama were selected from samples submitted to the State Federal Brucellosis Laboratory at Auburn, AL, between October 1985 and April 1986 (see Table 1). Samples submitted to the brucellosis surveillance system are from cattle that are being moved within or out of Alabama and therefore this was not a random sample of Alabama cattle. In order to study recent infections, only samples from cattle ~<2 years of age were tested. Samples were selected from sera submitted to the Brucellosis Laboratory during the 6-month period of the study.

Serological tests Sera were screened for group-specific antibodies to BTV using the agar gel precipitin (AGP) test described by Jochim (1976). Sera positive by the AGP test were tested for type-specific antibody using the serum neutralization (SN) test. Vero cells (MARU strain ) were grown in 96-well microtiter plates in MeTABLE 1 Results of agar gel precipitin ( A G P ) tests a n d bluetongue virus serotype 2 ( B T V - 2 ) serum neutralization from bovine serum samples collected from five regions of Alabama during 1985-1986 Region

Eastern coastal plain Western coastal plain Piedmont Eastern plateau Western plateau Total

Samples

Farms

A G P positive

BTV-2 positive I

Samples

Farms

Samples

Farms

178 278 80 598 207

65 93 32 168 75

65 116 26 176 63

37 50 17 101 39

3 5 5 12 3

3 3 4 11 3

1341

433

446

244

28

24

Samples in which serum neutralization titers to BTV-2 were 4-fold greater t h a n any titer to the other four serotypes.

115

dium 199 (Gibco Laboratories, Grand Island, NY) and 10% fetal bovine serum plus antibiotics (Microbiological Associates, Lenexa, Kansas). Initially, 25 ,ul of serum was diluted 1:10 in 25/~l of BTV (100 TCIDs0). Twofold dilutions were then made to a final dilution of 1 : 1280 in a single row of wells. Known positive and negative antisera to BTV were included with each test. The plates were incubated at 37 °C for 2 h. Following incubation, the virus-serum mixture was added to plates containing confluent layers of Vero cells, and these plates were incubated for 96 h. Titers were expressed as the reciprocal of the final dilution at which no cytopathic effect was observed. Samples positive for BTV2 antibody were then titered against the other four serotypes of BTV occurring in the U.S.A. The five virus serotypes used in this study were obtained from Dr. James Pearson, National Veterinary Service Laboratory, Ames, IA, 50010. RESULTS

A total of 1341 samples representing 433 farms in 65 of the 67 counties of Alabama were tested for antibodies to BTV (Table 1 ). Four hundred and forty-

WESCO.a,! PL~

Fig. 1. The presence of bluetongue virus serotype 2 (BTV-2) in five regions of Alabama. Hatched areas represent counties in which BTV-2 was identified serologically during 1985-1986. Data for the positive counties are represented as the number of farms positive for BTV-2/total number of farms sampled.

116 TABLE 2

Cross-reactivity of 84 bovine serum samples which tested positive for antibody to bluetongue virus serotype 2 (BTV-2) by serum neutralization in Alabama during 1985-1986. Some sera reacted to more than one BTV serotype Titer

to

BTV-2

Total samples

Titer to other BTV serotypes BTV-10 0

< 20 20-40 80-160 320-1000 > 1280

10 20

BTV-11 >40

0

10 20

BTV-13 >40

0

10 20

BTV-17 >40

0

10-20

40 160

>320

(17) (37) (12) (7) (11)

16 31 0 2 0

1 6 5 2 6

0 0 7 3 5

14 20 1 2 5

3 10 8 3 6

0 7 3 2 0

6 22 10 7 2

8 10 0 0 5

3 5 2 0 3

4 8 2 3 3

7 22 2 1 5

5 7 8 1 2

1 0 0 2 1

Total (84) % Total crossreactivity

49

20

15

42

30

12

47

23

13

20

37

23

4

42(35/84)

50(42/84)

43(36/84)

76(64/84)

TABLE 3

Serum neutralization titers of bluetongue virus serotype 2 ( B T V - 2 ) - p o s i t i v e bovine sera to bluet o n g u e virus serotypes 10, 11, 13 and 17 in 11 s a m p l e s that titered greater than 1280 to B T V - 2 Titer

B T V serotype 10

11

13

17

< 10 10- 20 40-80 160-320 640-1280

0 6 5 0 0

5 6 0 0 0

3 5 2 1 0

3 5 2 0 0

> 1280

0

0

0

1

six (446) of the 1341 samples (33%) were positive by the AGP test. These positive samples represented 244 of the 433 farms (56%) included in the study. Eighty-four of the 446 samples (19%) positive by AGP were positive for antibodies to BTV-2 at a 1:10 dilution or greater. When these samples were tested for antibodies to the other four serotypes of BTV, nine were positive only to B T V - 2 . An additional 19 samples had titers to BTV-2 that were 4-fold greater than any titer to the other four serotypes. Therefore, BTV-2 was confirmed serologically in 28 of the 84 samples (33%). The presence of BTV-2 was confirmed serologically in all five regions of Alabama (Table 1 ). Samples positive for BTV-2 were from 19 of 67 counties in Alabama (28%) (Fig. 1 ). The degree of cross-reactivity of the 84 samples that were positive for BTV2 is given in Table 2. The greatest cross-reactivity was to serotype 17 (76%).

117 Serotypes 10 and 13 had the lowest cross-reactivity rates (42 and 43%, respectively). Sixty-seven samples had a titer to BTV-2 of >/1:20. When the titers of these sera to the other four serotypes were determined, 15 (22%), 12 (18%), 10 (15%) and 21 (31%) had titers of >~1:40 to BTV-10, BTV-11, BTV-13 and BTV-17, respectively. The cross-reactivity of 11 samples that titered > 1280 to BTV-2 is shown in Table 3. Only two of the 11 samples had titers > 1:80 to the other serotypes of BTV. DISCUSSION Florida and Alabama have apparent differences in the epidemiology and patbogenesis of BTV-2. The virus was isolated from disease-free cattle in Florida, but the first isolations in Alabama were from clinically ill animals (Lauerman et al., 1986). Culicoides insignis was suspected to be the vector involved in the transmission of BTV-2 in Florida since an isolation of BTV-2 was made from a pool of these insects (Greiner et al., 1985). This species occurs only in extreme southern Alabama near the Florida border. Culicoides variipennis, which is considered the major vector of BTV in North America, was present at both BTV-2 outbreak sites in Alabama. Culicoides debilipalpis and C. steUifer were also collected in appreciable numbers during the outbreaks at both sites (Mullen and Lauerman, 1986). However, attempts to isolate BTV from these three field-collected species were unsuccessful. Serological data on BTV must be interpreted carefully (Carlson, 1983). A single serum may react with several BTV serotypes (Gorman, 1979; DellaPorta et al., 1983). In this study, 75 of 84 samples (89%) that were positive to BTV-2 cross-reacted with other serotypes. Individual animals may be infected with more than one type of BTV (Della-Porta et al., 1983). Natural infections with two serotypes of BTV occur in both cattle and sheep (Stott et al., 1982). Another consideration is cross-reactivity in animals infected with only one serotype. Cross-reactivity usually occurs in sera that have low titers to the virus (Della-Porta et al., 1983). The seropositivity rate of farms in this study (56%) agrees with a previous Alabama survey result of 52% (Haynes et al., 1982) indicating that infection with BTV is common throughout Alabama. Despite the widespread occurrence of BTV-infected cattle, only 19 of 50 Alabama veterinarians (38%), responding to a mailed survey, indicated that they had diagnosed bluetongue during 1985 (J.C. Wright, L.H. Lauerman, K.E. Nusbaum and G.R. Mullen, unpublished data, 1986). Diagnoses were most often made using clinical signs and serology. This suggests that BTV cases in Alabama are usually subclinical or go undiagnosed due to their similarity to other diseases. Serological evidence indicates that BTV-11 and -17 are the most prevalent serotypes in the south-eastern U.S.A. (Haynes et al., 1982 ). In this study, BTV2-positive sera cross-reacted most frequently with these serotypes. A lower, but substantial number of BTV-2-positive sera also reacted with serotypes 10

118 a n d 13. T h e p r e s e n c e of at least two, a n d p o s s i b l y m o r e , B T V s e r o t y p e s in A l a b a m a p r o v i d e s o p p o r t u n i t y for dual i n f e c t i o n s a n d genetic r e c o m b i n a t i o n to o c c u r i n c r e a s i n g t h e p r o b a b i l i t y of t h e p r o d u c t i o n of n e w s t r a i n s ( S t o t t et al., 1982, 1987). T h i s s t u d y d o c u m e n t s t h e p r e s e n c e of B T V - 2 in c o u n t i e s t h r o u g h o u t Alab a m a . Since t h e s a m p l e s u s e d in t h i s s u r v e y were n o t r a n d o m , h e r d risk f a c t o r s a n d B T V p r e v a l e n c e could n o t be d e t e r m i n e d . A d d i t i o n a l studies are n e e d e d to assess t h e i m p a c t of t h i s new s e r o t y p e on t h e livestock i n d u s t r y . F u r t h e r studies are n e e d e d in A l a b a m a to d e t e r m i n e t h e Culicoides species r e s p o n s i b l e for t h e t r a n s m i s s i o n of this s e r o t y p e of B T V a n d risk factors for B T V infection. ACKNOWLEDGEMENTS T h i s r e p o r t is b a s e d on w o r k s u p p o r t e d b y F o o d A n i m a l H e a l t h a n d D i s e a s e R e s e a r c h F u n d s ( P r o j e c t No. 2-12147), P u b l i c a t i o n No. 1945, College of Vete r i n a r y Medicine. T h e a u t h o r s t h a n k K a t h y D r i g g e r s a n d L i s a B a r t o l o t t o for technical assistance.

REFERENCES Barber, T.L., 1979. Temporal appearance, geographic distribution, and species of origin of bluetongue virus serotypes in the United States. Am. J. Vet. Res., 40: 1654-1656. Browne, J.G., 1971. Bluetongue disease. Adv. Vet. Sci. Comp. Med., 15: 1-46. Carlson, J.H., 1983. Differential serotyping of bluetongue virus-positive serums obtained in a national survey. In: R.W. Compans and D.H.L. Bishop (Editors), Double-Stranded RNA Viruses. Elsevier, New York, pp. 361-366. Della-Porta, A.J., Sellers, R.F., Herniman, K.A.J., Littlejohns, I.R., Cybinski, D.H., St. George, T.D., McPhee, D.A., Snowdan, W.A,, Campbell, J., Cargill, C., Corbould, A., Chung, Y.S. and Smith, V.W., 1983. Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses. Vet. Microbiol., 8: 147-162. Du Toit, R.M., 1944. The transmission of blue-tongue and horse-sickness by Culicoides. Ondestepoort J. Vet. Sci. Anim. Ind., 19: 7-16. Erasmus, B.J., 1975. Bluetongue in sheep and goats. Aust. Vet. J., 51: 165-170. Gibbs, E.P.J., Greiner, E.C., Taylor, W.P., Barber, T.L., House, J.A. and Pearson, J.E., 1983. Isolation of bluetongue virus serotype 2 from cattle in Florida: serotype of bluetongue virus hitherto unrecognized in the Western Hemisphere. Am. J. Vet. Res., 44: 2226-2228. Gorman, B.M., 1979. Variation in orbiviruses. J. Gen. Virol., 44: 1-15. Greiner, E.C., Barber, T.L., Pearson, J,E., Kramer, W.L. and Gibbs, E.P.J., 1985. Orbiviruses from Culicoides in Florida. In: T.L. Barber and M.M. Jochim (Editors), Bluetongue and Related Orbiviruses. Alan R. Liss, New York, pp. 195-200. Haynes, T.B., Harrold, S. and Schultz, R.D., 1982. A seroepidemiologic survey of antibody to bluetongue virus in Alabama cattle. Cornell Vet., 72: 262-268. Hourrigan, J.L. and Klingsporn, A.L., 1975. Bluetongue: the disease in cattle. Aust. Vet. J., 5l: 170-174.

119 House, J.A., Groocock, C.M. and Campbell, C.H., 1982. Antibodies to bluetongue viruses in animals imported into the United States zoological gardens. Can. J. Comp. Med., 46: 154-159. Jochim, M.M., 1976. Improvement of the AGP test for bluetongue. Proc. Am. Assoc. Vet. Lab. Diagn., 19: 361-376. Lauerman, L.H., Luedke, A.J., Barber, T.L., Mullen, G.R., Roy, P., Wright, J.C., Wolfe, D.E. and Nusbaum, K.E., 1986. Spread of bluetongue virus serotype 2 in the United States. In: P. Roy, C.E. Schore and B.I. 0sborn (Editors), Proc. Int. Symp. Double Stranded RNA Viruses. Oxford, Gt. Britain, pp. 1-6. Metcalf, H.E. and Luedke, A.J., 1980. Bluetongue and related disease. Bov. Pract., 15: 188-191. Mullen, G.R. and Lauerman, L.H., 1986. Bluetongue serotype 2 poses concern for Alabama cattlemen. Highlights Agric. Res., 33: 16. Osburn, B.I., 1983. Bluetongue and bovine leukosis committee report. Proc. U.S. Anim. Health Assoc., 87: 111-115. Stott, J.L., Osburn, B.I. and Barber, T.L., 1982. Recovery of dual serotypes of bluetongue virus from infected sheep and cattle. Vet. Microbiol., 7: 197-207. Stott, J.L., Osburn, B.I. and Oberst, R.D., 1987. Genetic diversity of bluetongue virus. Proc. U.S. Anim. Health Assoc., 91: 229-234.