African Swine Fever Virus Infection in the Soft Tick, Ornithodoros (Alectorohius) puertoricensis (Acari: Argasidae) R. G. ENDRIS,i T. M. HASLETT,2 AND W. R. HESS* Plum Island Animal Disease Center, USDA-ARS-NAA, P.O. 848, Greenport, NY 11944-0848
J. Med. Entomol. 29(6): 990-994 (1992)
KEY WORDS Ornithodoros (A.) puertoricensis, arbovirus, African swine fever virus
HOOGSTRAAL (1985) reported that 126 arboviruses have been recovered from an equal number of tick species. Forty-nine arboviruses have been reported from 34 argasid species, and the argasids act as either reservoirs or vectors for —40 of these viruses. To the best of our knowledge, there are no extant reports regarding the effects of arboviruses on tick vectors and particularly the argasid vectors. However, the effects of viral pathogens on mosquito vectors include cytopathic effects on the salivary glands (Mims et al. 1966), decreased survival after the infective blood meal (Turell et al. 1985, Faran et al. 1987), reduced refeeding rates (Grimstad et al. 1980, Turell et al. 1985), reduced fecundity (Tesh 1980, Turell et al. 1985), and increased development times (Beaty et al. 1980, Tesh 1980, Turell et al. 1982). Extensive investigations on the role of the African argasid Ornithodoros moubata Murray as a reservoir and vector for African swine fever virus (Plowright et al. 1969a, b, 1970; Grieg 1972) did not reveal untoward viral effects on the ticks, or, 1 Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065-0900. 2 792 Chimney Rock Road, Martinsville, NJ 08836. 3 395 Gillette Drive, East Marion, NY 11939.
at least, such effects were not reported. In conducting experimental investigations on the vector capacity of the Nearctic argasid Ornithodoros coriaceus Koch for African swine fever virus, (ASFV) Groocock et al. (1980) noted that 80% mortality occurred in nymphs fed on a viremic pig. Hess et al. (1989) reported that infection with ASFV caused mortality in adult female O. moubata and Ornithodoros erraticus (Lucas) (=Ornithodoros marocanus Velu). The effects of ASFV infection on the Neotropical argasid Ornithodoros (Alectorobius) puertoricensis Fox were studied as part of investigations on the vector capacity of the tick for ASFV (Endris et al. 1991a), and the results are reported here.
Materials and Methods
Swine, swine-infection methods, and methods for detection of ASFV in swine blood and ticks were described by Endris et al. [1991a]. Virus. An isolate taken from infected domestic swine in the Dominican Republic in 1979 (Mebus & Dardiri 1979) was used throughout this study. The isolate, named DR-II, was hemadsorbing and moderately virulent (Pan & Hess 1984). The isolate had been passed twice in
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
ABSTRACT In total, 1,186 second instar Ornithodoros (Alectorobius) puertoricensis Fox second instars were fed on a pig when it had a viremia of 105"2 hemadsorption units (HAd50/ml) and 420 second-instar O. puertoricensis were fed on an uninfected pig. Subsequent blood meals for ticks in both groups were from uninfected pigs. The effects of African swine fever virus (ASFV) infection on O. puertoricensis populations were evaluated for the following parameters: mortality; mean time to death; percentage molted per instar; percentage molted to male, female, or subsequent instar; effects on duration of premolt period; and the number of blood meals per instar. The cumulative virus-induced mortality rate for all immature stages (second to fifth instar) of O. puertoricensis that had been fed as second instars on a pig infected with ASFV was 43.2%. In contrast, 23.1% mortality was observed among ticks fed on uninfected pigs. The mortality rate among third instars that fed on the viremic pig was 55.3% versus 4.8% among nymphs fed on normal pigs. One-third to more than one-half of all third, fourth, and fifth instars required at least two blood meals to molt. Mean premolt periods for second, third, fourth, fifth, and sixth instars fed on uninfected pigs were =12, 15, 32, 22, and 14 d, respectively. Mean weights for unfed second to fifth instars, males, and females were: 0.6, 1.0, 1.5, 1.7, 1.5, and 3.1 mg per tick, respectively.
November 1992
ENDRIS ET AL.: AFRICAN SWINE FEVER VIRUS IN
Results Mortality rates for ticks fed as second instars on either viremic or uninfected pigs with all subsequent blood meals for both groups of ticks from uninfected pigs are shown in Table 1. There were no differences in mortality rates for second, fourth, fifth, and sixth instars. However, the mortality rate for third instars that had fed on a viremic pig as second instars was 55.3% compared with 4.8% mortality observed in nymphs that had
991
Table 1. Mortality rates for each instar of O. puertoricensis that had fed as second instars either on an ASFVinfected pig or on a normal pig No. instar 2 2 2 Total 3 3 3 3 Total 4 4 4 4 Total 5 5 5 Total 6 6 Total Total (life cycle)
DIOOu
meals" 1 2 3 1 2 3 4 1 2 3 4 1 2 3 1 2
Infected
Control 6
% Mortality*
14.1 (135/960) 30.0 (18/60) 20.0 (1/5) 16.0 (154/960) 30.6 (132/432) 46.2 (96/208) 24.3 (9/37) 28.6 (2/7) 55.3 (239/432) 8.4 (10/119) 9.2 (9/98) — — 16.0 (19/119) 14.3 (2/14) 9.1 (1/11) 0.0 (0/2) 21.4 (3/14) 0.0 (0/1) 0.0 (0/1) 0.0 (0/1) 43.2 (415/960)
18.1(76/420) 0.0 (0/6) — 18.1 (76/420) 3.7 (7/188) 3.4 (2/59) 0.0 (0/1) — 4.8 (9/188) 6.4 (4/62) 19.5 (8/41) 0.0 (0/7) 0.0 (0/1) 19.4 (12/62) 0.0 (0/4) 0.0 (0/2) 0.0 (0/2) 0.0 (0/4) 0.0 (0/1)
% Mortality
0.0 (0/1) 23.1 (97/420)
a After the first blood meal, all blood meals for nymphs in both groups were from uninfected pigs. b Numbers in parentheses indicate number dead/number fed.
fed on a normal pig. As a result of the high mortality rate for third instars, the overall mortality rate for all nymphs that had an infective blood meal was 43.2%, or nearly twice that observed (23.1%) in nymphs that fed only on uninfected pigs. The mean time to death for nymphs is shown in Table 2. Two general trends were evident from the patterns of mortality shown: (1) the time to death for ASFV-infected nymphs occurred over a broader range of time, especially for second and third instars and (2) infected nymphs tended to survive longer before dying than noninfected nymphs. The duration of the premolt period (time from blood meal to the next molt) for each nymphal instar of O. puertoricensis fed on pigs is shown in Table 3. Premolt periods for both infected and uninfected nymphs were nearly equal for each instar. Approximately 12, 15, 32, 22, and 14 d were required for fed second to sixth instars to molt, respectively. The majority of ticks molted within 30 d after feeding. The total duration of premolt periods for development from second instars to adults was 12—144 d for males and 26— 207 d for females. It should be noted that this is the first report of biological data for O. puertoricensis fed on pigs and reared under controlled conditions. Molting and eclosion patterns for ticks fed as second instars on either a viremic or an uninfected pig were similar (Table 4). Nearly all fed
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
swine and frozen in a spleen-blood suspension at — 70°C for 4 yr before this study. Ticks. O. puertoricensis (U.S. National Tick Collection, Georgia Southern University, Statesboro, GA, RML No. 117905) collected from Haiti were mass-reared at 27—28°C by the methods of Endris et al. (1986). None of the ticks collected from the Dominican Republic or Haiti was infected with ASFV; nevertheless, field collected ticks were shown to be virus-free by two methods. First, all ticks were fed on susceptible pigs, which were negative for ASFV both by attempted virus isolation from blood and by seroconversion. Second, all ticks used as parental stocks were individually assayed for virus after several gonotrophic cycles and were found to be negative. Pooled larvae from the first gonotrophic cycles were also negative for virus. Six days after it was infected, 1,186 second instar O. puertoricensis were fed on a pig when it had a viremia of 105 2 hemadsorption units (HAd50/ml), and 420 second instar O. puertoricensis were fed on an uninfected pig. Pigs were anesthetized with sodium thiopental (250 mg/ml) (Abbott Laboratories, Chicago, IL) given intraperitoneally at a dosage of 1 gm analgesic in 25% aqueous solution per 12 kg body weight. Ticks were transferred to a bottomless plastic container from which the lid had been removed/that was held on the shaved skin of the pig. After feeding to repletion, ticks were removed from the feeding container with a forceps as they detached. Subsequently, both groups of nymphs in each instar were fed on the same naive pig. Uninfected ticks were fed
J. Med. Entomol. 29(6): 990-994 (1992)
KEY WORDS Ornithodoros (A.) puertoricensis, arbovirus, African swine fever virus
HOOGSTRAAL (1985) reported that 126 arboviruses have been recovered from an equal number of tick species. Forty-nine arboviruses have been reported from 34 argasid species, and the argasids act as either reservoirs or vectors for —40 of these viruses. To the best of our knowledge, there are no extant reports regarding the effects of arboviruses on tick vectors and particularly the argasid vectors. However, the effects of viral pathogens on mosquito vectors include cytopathic effects on the salivary glands (Mims et al. 1966), decreased survival after the infective blood meal (Turell et al. 1985, Faran et al. 1987), reduced refeeding rates (Grimstad et al. 1980, Turell et al. 1985), reduced fecundity (Tesh 1980, Turell et al. 1985), and increased development times (Beaty et al. 1980, Tesh 1980, Turell et al. 1982). Extensive investigations on the role of the African argasid Ornithodoros moubata Murray as a reservoir and vector for African swine fever virus (Plowright et al. 1969a, b, 1970; Grieg 1972) did not reveal untoward viral effects on the ticks, or, 1 Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065-0900. 2 792 Chimney Rock Road, Martinsville, NJ 08836. 3 395 Gillette Drive, East Marion, NY 11939.
at least, such effects were not reported. In conducting experimental investigations on the vector capacity of the Nearctic argasid Ornithodoros coriaceus Koch for African swine fever virus, (ASFV) Groocock et al. (1980) noted that 80% mortality occurred in nymphs fed on a viremic pig. Hess et al. (1989) reported that infection with ASFV caused mortality in adult female O. moubata and Ornithodoros erraticus (Lucas) (=Ornithodoros marocanus Velu). The effects of ASFV infection on the Neotropical argasid Ornithodoros (Alectorobius) puertoricensis Fox were studied as part of investigations on the vector capacity of the tick for ASFV (Endris et al. 1991a), and the results are reported here.
Materials and Methods
Swine, swine-infection methods, and methods for detection of ASFV in swine blood and ticks were described by Endris et al. [1991a]. Virus. An isolate taken from infected domestic swine in the Dominican Republic in 1979 (Mebus & Dardiri 1979) was used throughout this study. The isolate, named DR-II, was hemadsorbing and moderately virulent (Pan & Hess 1984). The isolate had been passed twice in
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
ABSTRACT In total, 1,186 second instar Ornithodoros (Alectorobius) puertoricensis Fox second instars were fed on a pig when it had a viremia of 105"2 hemadsorption units (HAd50/ml) and 420 second-instar O. puertoricensis were fed on an uninfected pig. Subsequent blood meals for ticks in both groups were from uninfected pigs. The effects of African swine fever virus (ASFV) infection on O. puertoricensis populations were evaluated for the following parameters: mortality; mean time to death; percentage molted per instar; percentage molted to male, female, or subsequent instar; effects on duration of premolt period; and the number of blood meals per instar. The cumulative virus-induced mortality rate for all immature stages (second to fifth instar) of O. puertoricensis that had been fed as second instars on a pig infected with ASFV was 43.2%. In contrast, 23.1% mortality was observed among ticks fed on uninfected pigs. The mortality rate among third instars that fed on the viremic pig was 55.3% versus 4.8% among nymphs fed on normal pigs. One-third to more than one-half of all third, fourth, and fifth instars required at least two blood meals to molt. Mean premolt periods for second, third, fourth, fifth, and sixth instars fed on uninfected pigs were =12, 15, 32, 22, and 14 d, respectively. Mean weights for unfed second to fifth instars, males, and females were: 0.6, 1.0, 1.5, 1.7, 1.5, and 3.1 mg per tick, respectively.
November 1992
ENDRIS ET AL.: AFRICAN SWINE FEVER VIRUS IN
Results Mortality rates for ticks fed as second instars on either viremic or uninfected pigs with all subsequent blood meals for both groups of ticks from uninfected pigs are shown in Table 1. There were no differences in mortality rates for second, fourth, fifth, and sixth instars. However, the mortality rate for third instars that had fed on a viremic pig as second instars was 55.3% compared with 4.8% mortality observed in nymphs that had
991
Table 1. Mortality rates for each instar of O. puertoricensis that had fed as second instars either on an ASFVinfected pig or on a normal pig No. instar 2 2 2 Total 3 3 3 3 Total 4 4 4 4 Total 5 5 5 Total 6 6 Total Total (life cycle)
DIOOu
meals" 1 2 3 1 2 3 4 1 2 3 4 1 2 3 1 2
Infected
Control 6
% Mortality*
14.1 (135/960) 30.0 (18/60) 20.0 (1/5) 16.0 (154/960) 30.6 (132/432) 46.2 (96/208) 24.3 (9/37) 28.6 (2/7) 55.3 (239/432) 8.4 (10/119) 9.2 (9/98) — — 16.0 (19/119) 14.3 (2/14) 9.1 (1/11) 0.0 (0/2) 21.4 (3/14) 0.0 (0/1) 0.0 (0/1) 0.0 (0/1) 43.2 (415/960)
18.1(76/420) 0.0 (0/6) — 18.1 (76/420) 3.7 (7/188) 3.4 (2/59) 0.0 (0/1) — 4.8 (9/188) 6.4 (4/62) 19.5 (8/41) 0.0 (0/7) 0.0 (0/1) 19.4 (12/62) 0.0 (0/4) 0.0 (0/2) 0.0 (0/2) 0.0 (0/4) 0.0 (0/1)
% Mortality
0.0 (0/1) 23.1 (97/420)
a After the first blood meal, all blood meals for nymphs in both groups were from uninfected pigs. b Numbers in parentheses indicate number dead/number fed.
fed on a normal pig. As a result of the high mortality rate for third instars, the overall mortality rate for all nymphs that had an infective blood meal was 43.2%, or nearly twice that observed (23.1%) in nymphs that fed only on uninfected pigs. The mean time to death for nymphs is shown in Table 2. Two general trends were evident from the patterns of mortality shown: (1) the time to death for ASFV-infected nymphs occurred over a broader range of time, especially for second and third instars and (2) infected nymphs tended to survive longer before dying than noninfected nymphs. The duration of the premolt period (time from blood meal to the next molt) for each nymphal instar of O. puertoricensis fed on pigs is shown in Table 3. Premolt periods for both infected and uninfected nymphs were nearly equal for each instar. Approximately 12, 15, 32, 22, and 14 d were required for fed second to sixth instars to molt, respectively. The majority of ticks molted within 30 d after feeding. The total duration of premolt periods for development from second instars to adults was 12—144 d for males and 26— 207 d for females. It should be noted that this is the first report of biological data for O. puertoricensis fed on pigs and reared under controlled conditions. Molting and eclosion patterns for ticks fed as second instars on either a viremic or an uninfected pig were similar (Table 4). Nearly all fed
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
swine and frozen in a spleen-blood suspension at — 70°C for 4 yr before this study. Ticks. O. puertoricensis (U.S. National Tick Collection, Georgia Southern University, Statesboro, GA, RML No. 117905) collected from Haiti were mass-reared at 27—28°C by the methods of Endris et al. (1986). None of the ticks collected from the Dominican Republic or Haiti was infected with ASFV; nevertheless, field collected ticks were shown to be virus-free by two methods. First, all ticks were fed on susceptible pigs, which were negative for ASFV both by attempted virus isolation from blood and by seroconversion. Second, all ticks used as parental stocks were individually assayed for virus after several gonotrophic cycles and were found to be negative. Pooled larvae from the first gonotrophic cycles were also negative for virus. Six days after it was infected, 1,186 second instar O. puertoricensis were fed on a pig when it had a viremia of 105 2 hemadsorption units (HAd50/ml), and 420 second instar O. puertoricensis were fed on an uninfected pig. Pigs were anesthetized with sodium thiopental (250 mg/ml) (Abbott Laboratories, Chicago, IL) given intraperitoneally at a dosage of 1 gm analgesic in 25% aqueous solution per 12 kg body weight. Ticks were transferred to a bottomless plastic container from which the lid had been removed/that was held on the shaved skin of the pig. After feeding to repletion, ticks were removed from the feeding container with a forceps as they detached. Subsequently, both groups of nymphs in each instar were fed on the same naive pig. Uninfected ticks were fed
