Transovarial transmission of Rio Grande virus (Bunyaviridae: Phlebovirus) by the sand fly, Lutzomyia anthophora.

The growth of Rio Grande (RG) virus, the only phlebovirus known to occur in the United States, was studied in Lutzomyia anthophora, its suspected sand fly vector. RG viral titers in infected flies increased more than 10,000-fold within 7 days after intrathoracic inoculation. Experimentally infected female L. anthophora transmitted virus transovarially (vertically) to 54.8% of their F1 progeny. This is the first virologically confirmed demonstration of transovarial transmission of a phlebovirus by sand flies. It indicates one mechanism by which RG and possibly other phleboviruses may be maintained in nature.

Identification of African swine fever viral antigens in the hemolymph of soft ticks (Argasidae: Ornithodoros) by the immunodot blot test.

The immunodot blot test was used to identify African swine fever virus (ASFV) antigens in the hemolymph from soft ticks (Ornithodoros coriaceus) fed on ASFV-infected pigs. The immunodot blot test was sensitive and specific for ASFV antigens and has potential field application. Hemolymph from field-collected ticks can be screened for ASFV and a variety of other tick-borne pathogens using this test.

A mouse lethal dose assay for detection and titration of Cowdria ruminantium (Kwanyanga strain) in goats and ticks.

A mouse lethal dose assay was used to detect a mouse pathogenic strain (Kwanyanga) of Cowdria ruminantium, the etiological agent of heartwater in goats and ticks. The titer of the rickettsial organisms in goat blood was directly related to the febrile response of the goat and the rickettsia were undetectable after the fever subsided. The maximum rickettsial titer in goat blood was 10(3) mouse LD50 ml-1. Cowdria-infected goat blood was shown to retain infectivity when held on ice for up to 2 h, but when held at room temperature infectivity declined by greater than 50% in 2 h. The mouse assay detected Cowdria in feeding female Amblyomma variegatum only on the eighth day of feeding and in feeding males on the second and eleventh days of feeding. Cowdria was shown to persist in the hemolymph of the soft tick Ornithodoros coriaceus for a period of at least 2 years.

Attempted transovarial and venereal transmission of African swine fever virus by the Iberian soft tick Ornithodoros (Pavlovskyella) marocanus (Acari: Ixodoidea: Argasidae).

Transovarial transmission experiments were conducted with three groups of Ornithodoros (Pavlovskyella) marocanus Velu; one group consisted of 27 pairs of adults that had been fed as larvae on a pig with a viremia of 10(7.4) HAd50/ml of African swine fever virus (ASFV). The second and third groups each consisted of 100 pairs of adults fed on a viremic pig (10(4.5) HAd50/ml) as adults. The first group underwent five gonotrophic cycles over a 554-d period. The second and third groups underwent three and two gonotrophic cycles, respectively. All larvae were fed in individual cohorts on naive pigs and the resulting nymphs were assayed by cohort for ASFV. None of the larvae transmitted ASFV to naive pigs by bite and ASFV was not isolated in swine buffy coat cultures from any cohort of nymphs. Therefore, O. marocanus does not exhibit transovarial transmission of ASFV. Venereal transmission experiments were conducted with pairs of O. marocanus in which either the female (100 pairs) or the male (100 pairs), respectively, had fed on a viremic pig (10(4.5) HAd50/ml). Both groups underwent at least two gonotrophic cycles over a 470-d period, were sampled periodically for the presence of ASFV, and the progeny were tested for the presence of ASFV. Venereal transmission from male to female occurred in 10% (1/10) of O. marocanus after the first gonotrophic cycle, but not after the second or third gonotrophic cycle, and transovarian transmission in these groups was not observed. Venereal transmission from infected females to uninfected males did not occur. ASFV persisted through five gonotrophic cycles over a 554-d period in 30% of adults fed on a viremic pig as larvae. ASFV was cleared during three gonotrophic cycles within a year from nearly all ticks fed on a viremic pig as adults. Virus-induced mortality rats of 12-80% occurred among ticks fed on viremic animals, whereas, no mortality was seen in ticks fed on uninfected animals. ASFV infection in ticks did not effect feeding frequency, egg-hatch rate, or the oviposition rate among females fed on a viremic pig as adults. The oviposition rate for females fed on a viremic pig as larvae was reduced by 63.4%. Parthenogenesis was not observed among O. marocanus. The mean gonotrophic cycle duration for pig-fed O. marocanus at 27 degree C was 19.8 d and the mean fecundity was 88.3 +/- 17.9 eggs/female/gonotrophic cycle.

Experimental transmission of African swine fever virus by the soft tick Ornithodoros (Pavlovskyella) marocanus (Acari: Ixodoidea: Argasidae).

A total of 1,600 Ornithodoros (Pavlovskyella) marcocanus larvae were fed on a pig with a viremia of 10(7.4) HAd50/ml of African swine fever virus (ASFV). Infected larvae were sampled daily for 15 d, and nymphs were sampled at least once per instar until they became adults. Initial titers of 10(4.48) HAd50 per larva declined to 10(4.04) within 2 d. Larval titers reached a maximum of 10(6.0) HAd50 per larva 10 d after the infective blood meal. Nymphs of each instar were fed on a susceptible pig and in each case transmitted ASFV by bite. Virus titers for first to fourth instars ranged from 10(4.61) to 10(3.34) HAd50 per nymph. Transstadial survival occurred in subsequent first, second, third, and fourth instars with an 89% survival rate over 250 d. Approximately 30% of adult ticks that were infected as larvae remained infected and transmitted ASFV to susceptible pigs 588 d later. In addition, ASFV was recovered from the same adult ticks 655 d after the infective blood meal.

Experimental transmission of African swine fever virus by the tick Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae).

The soft tick Ornithodoros puertoricensis Fox has been found on the Caribbean island of Hispaniola (Haiti and the Dominican Republic) where African swine fever (ASF) was endemic from 1978 to 1984. To evaluate the vector potential of O. puertoricensis for African swine fever virus (ASFV), second-instar nymphs were experimentally infected by feeding on a viremic pig that was infected with the Dominican Republic isolate (DR-II) of ASFV. Subsequent infection rates and mean virus titers for individually triturated ticks were: second-instar nymph (95.4%, 10(4.38 +/- 0.85)), third-instar nymph (48.9%, 10(4.59 +/- 0.61)), male (46.7%, 10(4.36 +/- 0.61)), and female (35.3%, 10(4.38 +/- 1.09)). Infected ticks transmitted ASFV to susceptible pigs by bite 23, 85, 126, 160, 182, and 239 d after the infective blood meal. These findings show that ASFV is passed transstadially among O. puertoricensis and that O. puertoricensis can be an efficient vector of African swine fever virus.

African swine fever virus infection in the Iberian soft tick, Ornithodoros (Pavlovskyella) marocanus (Acari: Argasidae).

One thousand six hundred Ornithodoros (Pavlovskyella) marocanus Velu larvae were fed on a pig infected with African swine fever virus (titer: 10(7.4) HAd50/ml), and 1,600 larvae were fed on an uninfected pig. Ticks in each group were compared for mortality rates, mean time to death for ticks that died, mean time from feeding to either molting or eclosion, percentage of ticks that eclosed or molted, and the number of blood meals per nymph or instar. Cumulative virus-induced mortality for all immature stages (larvae to adult) of O. marocanus that had been fed as larvae on a pig infected with African swine fever was ca. 73% over a 390-d period. In contrast, less than 9% mortality was observed among ticks fed on uninfected pigs. Mean time to death for infected ticks was 15-87 d versus 10-17 d for uninfected ticks. Differences in the premolt period (number of days from blood meal to molt) between infected and control ticks were not observed. Mean premolt periods for larvae and first-, second-, third-, fourth-, and fifth-instar nymphs fed on pigs were 7, 9, 15, 11, 15, and 15 d, respectively. The majority of infected and all uninfected ticks required only one blood meal from pigs to molt. Mean weights for unfed second-, third-, and fourth-instar nymphs and males and females were 0.50, 0.67, 3.07, 3.63, and 5.91 mg, respectively.

African swine fever virus infection in the soft tick, Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae).

In total, 1,186 second instar Ornithodoros (Alectorobius) puertoricensis Fox second instars were fed on a pig when it had a viremia of 10(5.2) 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 approximately 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.

Clearance of African swine fever virus from infected tick (Acari) colonies.

Three laboratory colonies of the argasid tick Onithodoros moubata porcinus van der Merwe were started from collections made in 1983 at three different sites in Zimbabwe. All of the colonies contained ticks infected with African swine fever (ASF) virus that was readily transmitted by bite to domestic pigs. Although they were maintained on virus-free pig blood, ASF virus infections persisted in the colonies for at least 1 yr. Despite the fact that ASF virus passes transstadially, sexually, and transovarially in this tick species sometime during the following year, the virus disappeared from the colonies. Studies comparing fecundity in infected and uninfected lots of O. moubata porcinus showed that mortality rates were considerably higher among the infected ticks. A similar study with Ornithodoros erraticus Lucas, a tick that harbors and transmits ASF virus on the Iberian Peninsula, gave essentially the same results. This is probably a factor involved in the clearance of ASF virus from tick populations that are not subjected to reinfection. How this information may be applied in the eradication of African swine fever in Portugal and Spain is discussed.

Laboratory biology of Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae).

The laboratory biology of Ornithodoros (Alectorobius) puertoricensis Fox was studied over a 2-yr period. Approximately 100-150 ticks were reared individually at each of four temperatures: 22, 27, 33, and 40 degrees C and 90-95% RH. The mean egg incubation periods at those temperatures were 20.3, 11.1, 7.3, and 6.1 d, respectively. The average larval feeding period was 5.8 +/- 1.5 d for 15,875 larvae that fed on guinea pigs. The development times for first to fourth nymphal instars were as follows: 11.7, 48.5, 75.1, and 92.1 d, respectively, at 22 degrees C; 5.8, 18.8, 38.0, and 36.0 d, respectively, at 27 degrees C; 4.2, 10.5, 14.9, and 38.1 d, respectively, at 33 degrees C; and 5.8, 10.7, 21.2, and 35.3 d, respectively, at 40 degrees C. Males usually eclosed after three or four molts, and females usually eclosed after four or five molts. Approximately 10% of all nymphs required more than one blood meal per instar at least once during development. Twenty pairs of adults were held at each of three temperatures (22, 27, and 33 degrees C) for a year to study reproductive behavior. The number of gonotrophic cycles per female per year was 6.9, 9.8, and 10.8 at 22, 27, and 33 degrees C, respectively. The mean duration of the gonotrophic cycle was 42.3 d at 22 degrees C, 25.5 d at 27 degrees C, and 20.5 d at 33 degrees C. Mean egg production per female per gonotrophic cycle was 151 at 22 degrees C, 117 at 27 degrees C, and 130 at 33 degrees C and was not affected by temperature. O. puertoricensis did not exhibit autogeny or parthenogenesis. Hyperparasitism was observed in immatures and adults.

Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae): redescription by scanning electron microscopy.

The female, male, nymphal instars, and larva of Ornithodoros puertoricensis Fox are redescribed from specimens collected in Haiti. Data on host species and geographic distribution are also presented.

A further comparison of the thumb-counting and comb-counting techniques used to determine Ctenocephalides felis infestation levels on dogs.

A comparison was made to determine whether thumb-counting or comb-counting was more accurate for determining flea infestation levels on dogs when performed for equal periods of time. To accomplish this, ten beagle dogs were each infested with 100 adult fleas, Ctenocephalides felis. After the fleas were allowed to disperse for 1 h the dogs were examined using the thumb-counting method. The time required to cover each dog and the number of fleas counted were recorded. Thumb-counting times ranged from 3.0 to 4.8 min. Each of the dogs was then examined by the comb-counting method for the same amount of time it had been thumb-counted. The thumb-counting method detected a geometric mean of five (range, 0-13) fleas per dog, while comb-counting recovered a mean of 73.5 (range, 57-87) fleas per dog. These results were significantly different (P < 0.01), indicating that the differences in accuracy previously recorded for the two methods are independent of time. The standard deviations for both methods were also statistically significantly different, suggesting that comb-counting is also more precise than the thumb-counting method.

Effect of combing time on cat flea (Ctenocephalides felis) recovery from dogs.

Combing the haircoat to count fleas has been used to determine the efficacy of insecticides against fleas on dogs, but no standardization of method has been reported. In this study, the effect of combing time on flea recovery from dogs was examined. Six beagle dogs were infested with 100 unfed, adult Ctenocephalides felis (Bouché) on each of three consecutive days. A crossover design, balanced for carryover effects, was used to evaluate flea removal rates from each dog by comb-counting for three different time intervals; i.e. 5, 10 and 15 min. Each dog was combed once at each time interval on a different day, over three consecutive days. The results showed that the majority of fleas were recovered in the first 5 min of combing and there were no significant differences (P > or = 0.19) in the total number of fleas recovered between the 5, 10 or 15 min protocols. Moreover, the standard deviation and coefficient of variation increased with an increase in the amount of time spent combing, resulting in a decrease in precision for the longer protocols. Therefore, the comb time of 5 min provided a precise and accurate representation of the number of fleas present on an animal and could be useful as a standard measure of flea infestation levels in efficacy trials.

Potential arthropod vectors of African swine fever virus in North America and the Caribbean basin.

In an effort to identify arthropods that might serve as vectors and perhaps reservoirs of African swine fever virus (ASFV) if it were to enter the U.S.A., the blood-sucking insect Triatoma gerstaeckeri and four species of ticks of the genus Ornithodoros were established in colonies capable of reproducing in numbers sufficient to enable thorough studies to be made of their ASFV vector potentials. A nymphal stage of T. gerstaeckeri carried the virus for 41 days and retained it through one molt, but was unable to transmit it to susceptible pigs. Studies on O. coriaceus revealed that the species is able to harbor and transmit the virus for greater than 440 days, passing it trans-stadially from the first nymphal stage to the adult, sustaining it through at least four molts. Trans-ovarial passage was not demonstrated and nearly 40% of the ticks died, apparently, of the ASFV infection. O. turicata collected in Florida was also found to be capable of becoming infected with ASFV and transmitting it by bite to susceptible pigs. O. puertoricensis collected during the ASF eradication programs in the Dominican Republic and Haiti was not only readily infected experimentally, but it was also able to transmit the virus trans-stadially and trans-ovarially. However, ASFV was not isolated from any of the 350 O. puertoricensis collected in the Dominican Republic and Haiti. O. parkeri from a long-established laboratory colony were able to carry the virus through at least one molt, but they were unable to transmit it to susceptible pigs.

Comparison of thumb-counting and comb-counting methods to determine Ctenocephalides felis infestation levels on dogs.

Comb-counting and thumb-counting were compared in a cross-over study to determine which was more accurate for quantifying flea infestation levels on dogs. Twenty beagle dogs were used in the study and infested with either 50 or 100 adult fleas (Ctenocephalides felis). Two groups of five dogs each were infested with either 50 or 100 fleas per dog, and then comb-counted with a fine-toothed flea comb for 8 min periods. An additional two groups of five dogs each were also given 50 or 100 fleas, and then thumb-counted. The counting time for this technique is both lower and more variable because the fleas are only observed and not captured; thus, the speed at which the dog is covered must be increased in order to prevent counting the same fleas more than once. The mean time of thumb-counting per dog was 3.2 min. Fleas removed during comb-counting were placed back on the dog they were taken from after the count was concluded. At the cross-over point, the ten dogs that had been comb-counted were then thumb-counted and the ten dogs that had been thumb-counted were comb-counted. The results showed that comb-counting recovered significantly (P < or = 0.05) more fleas than did thumb-counting. On dogs given 50 and 100 fleas, comb-counting gave mean percentage recoveries of 67.6% and 75.4%, respectively, whereas thumb-counting found means of 8.8% and 7.7%, respectively. The order in which the counting methods were employed produced no significant effect (P > 0.05) on the number of fleas counted.

Detection and quantification of Ornithodoros-specific anti-tick antibody by competitive inhibition ELISA.

The objective of this study was to develop a highly specific enzyme-linked immunosorbent assay (ELISA) for the serological detection of anti-Ornithodoros tick antibodies in animals. Affinity-purified rabbit anti-Ornithodoros IgG antibodies were employed in indirect competitive inhibition ELISA assays designed to measure the anti-Ornithodoros antibody titers in other animal species using the domestic goat (Capra hircus) as a large animal model. Repeated infestation of goats with Ornithodoros coriaceus was found to elicit the formation of antibodies capable of inhibiting the binding of the Ornithodoros-specific rabbit IgG. Western blot analysis of goat and rabbit anti-tick antisera demonstrated both animal species to respond immunologically to a set of 9 major protein bands in O. coriaceus salivary gland extracts. The results of these experiments demonstrate that a history of animal exposure to O. coriaceus may be detected serologically by competitive inhibition ELISA.

Comparison of diluents for maintaining the viability of Cowdria ruminantium.

Thirteen solutions were compared to determine the optimal diluent for preservation of the viability of the Kwanyanga isolate of Cowdria ruminantium. They included the 2 diluents commonly used with C ruminantium and diluents proved effective with other rickettsiae. The capability of each diluent to maintain the viability of C ruminantium over a 3-hour period at room temperature was assessed by comparing the survival distributions of groups of outbred albino mice after they were inoculated IV with infected liver homogenates. The results indicated that the Snyder I diluent was significantly better at maintaining the viability of C ruminantium than were the other diluents studied.

Repellency and efficacy of 65% permethrin and 9.7% fipronil against Ixodes ricinus.

Two topically applied spot-on products--65% permethrin (Defend Exspot Treatment for Dogs, Schering-Plough Animal Health, Union, NJ) and 9.7% fipronil (Frontline Spot On Dog, Merial Limited, Iselin, NJ)--used for canine flea and tick control were evaluated for repellency against Ixodes ricinus, the tick species that is the primary vector of Lyme disease in Europe. Eighteen dogs were randomly assigned to the following treatment groups (n = 6): (1) 65% permethrin, (2) 9.7% fipronil, or (3) untreated control. Dogs were exposed to ticks in individually assigned cages with a carpet that covered =70% of the cage bottom. Dogs in treatment groups 1 and 2 were treated in accordance with label directions for each re-spective product on study day 0. Fifty unfed, male and female adult ticks were placed in the cages 15 to 30 minutes before the dogs. The dogs were placed in the cages for a 2-hour exposure period at 2, 7, 14, 21, 28, 35, and 41 days after treatment. After a 2-hour exposure period, dogs were removed from the cages and live (attached and unattached) and dead ticks were counted on the dogs, on the carpets, and in the cages. Cages were thoroughly cleaned and new carpet was used for each tick exposure period. Treatment of dogs with 65% permethrin reduced tick numbers on dogs by 99.1% at 2 days, 99.0% at 1 week, 95.9% at 2 weeks, 88.5% at 3 weeks, 87.1% at 4 weeks, and 48.0% at 6 weeks after application. In contrast, treatment of dogs with 9.7% fipronil reduced tick numbers on dogs by 61.4% at 2 days, 51.6% at 1 week, 37.0% at 2 weeks, 33.7% at 3 weeks, 10.8% at 4 weeks, and 0% at 6 weeks after application. The efficacy of 65% permethrin was significant (P < or = .05) when compared to the control at all challenges, whereas the efficacy of 9.7% fipronil was not significant as compared to controls at 21, 28, 35, and 41 days after treatment. The 65% permethrin killed significantly more Ixodes ricinus ticks (P < or = .05) than 9.7% fipronil from 2 to 41 days after treatment. The 65% permethrin repelled 1.9-, 2.0-, 3.0-, 43.3-, 3.9-, 8.9-, and 17.3-fold more Ixodes ricinus than did 9.7% fipronil at 2, 7, 14, 21, 28, 35, and 41 days after treatment, respectively, and all differences in repellency were significant (P < or = .05).

Efficacy of a topical spot-on containing 65% permethrin against the dog louse, Trichodectes canis (Mallophaga:Trichodectidae).

The efficacy of a 65% permethrin spot-on formulation (Defend EXspot, Schering-Plough Animal Health Corp., Union, NJ) against the dog louse, Trichodectes canis de Greer 1778, was studied. Fourteen dogs naturally infested with T. canis were evenly and randomly allocated to treatment with 65% permethrin administered at the label dose rate of 1 or 2 ml per dog or to an untreated control group. Louse counts were performed for each dog by gently back-combing the hair at six designated anatomic sites (head, tail, belly, each side, and an 8-cm strip the length of the body on the back), and lice were counted without removal on Days 0 (pretreatment), 7, 14, 21, and 28. Lice were eliminated from all dogs treated with the 65% permethrin spot-on within 7 days after treatment, and no subsequent reinfestations due to hatching of eggs were observed during the 28-day evaluation period. Untreated control dogs were subsequently treated with the 65% permethrin spot-on after the initial phase was completed and lice populations were evaluated as previously described. All lice were cleared from these dogs by Day 7, and there were no signs of reinfestation. No adverse reactions to treatment were noted during the study.

Efficacy of 65% permethrin applied as a topical spot-on against walking dandruff caused by the mite, Cheyletiella yasguri in dogs.

The efficacy of a 65% permethrin topically applied spot-on formulation (Defend EXspot Topical Remedy for Dogs, Schering-Plough Animal Health, Union, NJ) was determined against the dog mite, Cheyletiella yasguri (Smiley, 1965). Female dogs and their litters comprised the experimental unit, and all dogs in an experimental unit were treated on the same day 4 to 6 weeks after whelping. Mites and mite eggs were counted weekly on an untreated control group of six litters (15 pups) and on a group of six litters (14 pups) treated with 65% permethrin. Pups in the untreated control group maintained high numbers of Cheyletiella yasguri throughout the 14- to 21-day observation period. No mites or mite eggs were detected on dogs within 7 to 21 days after application of 65% permethrin. No adverse reactions were noted during the study. Clinical signs of infestation with C. yasguri--which included skin irritation, thickening of the stratum corneum, scratching with resultant scabs, pruritus, and flaky, scaly skin-were eliminated when mites were killed by the 65% permethrin formulation.