Bacteria associated with Amblyomma cajennense tick eggs

Ticks represent a large group of pathogen vectors that blood feed on a diversity of hosts. In the Americas, the Ixodidae ticks Amblyomma cajennense are responsible for severe impact on livestock and public health. In the present work, we present the isolation and molecular identification of a group of culturable bacteria associated with A. cajennense eggs from females sampled in distinct geographical sites in southeastern Brazil. Additional comparative analysis of the culturable bacteria from Anocentor nitens, Rhipicephalus sanguineus and Ixodes scapularis tick eggs were also performed. 16S rRNA gene sequence analyses identified 17 different bacterial types identified as Serratia marcescens, Stenotrophomonas maltophilia, Pseudomonas fluorescens, Enterobacter spp., Micrococcus luteus, Ochrobactrum anthropi, Bacillus cereus and Staphylococcus spp., distributed in 12 phylogroups. Staphylococcus spp., especially S. sciuri, was the most prevalent bacteria associated with A. cajennense eggs, occurring in 65% of the samples and also frequently observed infecting A. nitens eggs. S. maltophilia, S. marcescens and B. cereus occurred infecting eggs derived from specific sampling sites, but in all cases rising almost as pure cultures from infected A. cajennense eggs. The potential role of these bacterial associations is discussed and they possibly represent new targets for biological control strategies of ticks and tick borne diseases.     

The effects of the bont tick, Amblyomma hebraeum, on milk production of Sanga and Sanga x Brahman cattle

Abstract. The effects of adults of the bont tick, Amblyomma hebraeum on the milk production of Sanga and Sanga x zebu (Brahman) cattle were measured over a period of 11 weeks in the lowveld of Zimbabwe in the summer of 1986. Four groups of lactating cows, consisting of two breeds, each divided into a high and low tick treatment, were exposed to very low or high challenges of ticks and their milk production measured by weighing their calves before and after suckling. The liveweight gains (LWG) of the calves were also measured.
Tick burdens on the infested groups averaged around fourteen engorging females of A.hebraeum per day, which amounted to infestations of about 150 adult ticks. That is greater than most observed field infestations. This caused no significant reduction in milk yield or calf growth over the whole period, provided the teats of the dams had not been damaged by ticks. Mismothering occurred when teats were damaged. No breed differences were observed so all data was pooled for further analysis. Average calf LWGs of the high tick groups were reduced by 2.2kg (P < 0.01) during one 4-week period but overall the 3.9 kg difference in LWG of the tick treatment groups was not quite significant (P < 0.10). Although there was a poor relationship between tick numbers and reduced milk yield or calf LWG, the effects were always in the direction expected. The effects averaged 6 ± lOg reduction of milk and 2.6 ± 1.8g loss of LWG of calves for every female tick that engorged. It was concluded that milk production is not an important consideration when estimating the losses in production caused by A.hebraeum on Brahman x Sanga or Sanga breeds of cattle. Losses due to teat or udder damage could be much more important and need to be quantified.     

Distributions of the vectors of heartwater, Amblyomma hebraeum and Amblyomma variegatum (Acari: Ixodidae), in Zimbabwe

The tick vectors of heartwater (Cowdria ruminantium infection) in Zimbabwe, Amblyomma hebraeum and Amblyomma variegatum, historically were believed to be confined to the low-lying regions of the south and north-west of the country. However, country-wide surveys performed in 19751980 and 19881991 demonstrated that both species were also established in western parts of the highveld plateau and had started to encroach on the predominantly heartwater-free central and eastern highveld regions. To determine the current distributions of both the vectors and evaluate the potential threat of heartwater to animals in the highveld, a survey of ticks infesting cattle was performed in 1996 at 2994 locations in small-holder and large-scale commercial farming areas throughout Zimbabwe. Amblyomma hebraeum was collected at 1329 locations, A. variegatum at 72 locations and both A. hebraeum and A. variegatum at 13 locations. The results demonstrated that A. hebraeum was present, as previously recorded, throughout the southern half of the country and appeared to have undergone further limited spread into the central and eastern highveld regions. Only the northern-most region of the country appeared to be free of this species. Amblyomma variegatum was collected mainly in the north-west, as previously recorded, but was also found at isolated locations across the central highveld region and along the eastern border with Mozambique. This species was, however, still absent from the southern half and the northern-most regions of the country. An overlap of the distributions of the two species existed within a zone along the southern and eastern regions of the distribution of A. variegatum. These results suggest that the vectors of heartwater are spreading and threaten to introduce heartwater into intensive livestock-producing regions of the country.Exp Appl Acarol 22: 725740 © 1998 Kluwer Academic Publishers     

Ecdysteroids in the developing eggs of the desert locust, Schistocerca gregaria

Ecdysteroid levels in the developing eggs of Schistocerca gregaria were determined, at daily intervals, using gas chromatography and electron capture detection of ecdysteroid derivatives. Ecdysone and 20-hydroxyecdysone were present both as the free ecdysteroid and as polar conjugates. Total ecdysteroids reached a maximum of 40 ng/egg with ecdysone contributing the greater part.     

Ecdysteroids in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest

In order to understand the composition and quantitative variation of ecdysteriods in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest, we analyzed the ecdysteroid composition and titers in the adult and egg of this pest. On day 4 after eclosion, the titer of ecdysteriods in the male adult was 0.080 ng/adult, much lower than 5.978 ng/adult in the female adult. During the development of ovaries, the titer of ecdysteriods was low on the first two days, and high in the late period, with the peak (10.48 ng/ovary) appearing on day 3. During the development of eggs, the titer of ecdysteriods was about 0.010 ng/egg from day 1 to day 3, and then decreased to 0.006 ng/egg on day 4. In both adults and eggs, three main components of ecdysteriods were found by identification of HPLC/RIA. They were 20-hydroxyecdysone, 26-hydroxyecdysone, and an unidentified component.     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.     

Efficacy of pheromone-acaricide-impregnated tail-tag decoys for controlling the bont tick,Amblyomma hebraeum (Acari: Ixodidae), on cattle in Zimbabwe

A large-scale field test using pheromone-acaricide-impregnated plastic tail-tag decoys demonstrated excellent efficacy of these devices for control of the bont tick,Amblyomma hebraeum, on cattle in Zimbabwe. The tail tags were impregnated with a mixture containingo-nitrophenol, methyl salicylate, 2,6-dichlorophenol and phenylacetaldehyde and one of three different acaricides (cyfluthrin, flumethrin or alphacypermethrin).o-Nitrophenol and methyl salicylate are components of theA. hebraeum attraction-aggregation-attachment pheromone, while 2,6-dichlorophenol and phenylacetaldehyde are proven attractants for this tick. Botho-nitrophenol and methyl salicylate were lost gradually from the tags over 12 and 14 week periods, respectively. In field trials, tick counts were compared between cattle that received tail tags either impregnated with pheromone mixture alone, cyfluthrin and pheromone mixture, flumethrin and pheromone mixture, alphacypermethrin and pheromone mixture or were left untreated. During the first 3 month trial period, control of adult bont ticks was 94.9% with cyfluthrin tail tags and 87.5% with flumethrin tail tags. In general, there was no significant difference in bont tick numbers on cattle without tags and those with tail tags containing pheromone only. When the trial was repeated for another 3 month period, control of bont ticks with tail tags containing cyfluthrin and flumethrin was 99.3 and 95.1%, respectively. However, control of bont ticks using alphacypermethrin was only 79.2%. Overall, retention of tail tags was excellent although some loss was encountered during the rainy season. In addition to controlling bont ticks, the tail tags provided moderate control of other tick species (Rhipicephalus evertsi evertsi, Rhipicephalus zambeziensis andHyalomma spp.) simultaneously infesting cattle in the trials.Deceased.     

Attachment kinetics of the adult tick Amblyomma variegatum to cattle

At the beginning of the 1999 rainy season, three traditional cattle herds were monitored for 48 days while grazing in the bushy savannah of southwestern Burkina Faso. Cattle in each herd were caught on several occasions each day and the attached ticks were counted. This confirmed that Amblyomma variegatum Fabricius (Acari: Ixodidae) adults picked up in the pastures mainly attach to the interdigital areas (87% of the 791 ticks captured), and reach the predilection sites later (chest and udder/inguinal area) when the animals lie down. As many females as males attached to the hosts, but the seasonal distribution was very heterogeneous, with only a few females attaching as long as the humidity rate remained low. It is suggested that this prevents eggs from being laid when conditions are not optimal for their survival and that of the larvae. Ticks attached all day but the number picked up hourly and daily varied greatly according to their density on the pasture. As a general trend, confirmed by another study carried out in 2005, the number of ticks picked up daily increased from less than one tick/animal/day, before the onset of the rainy season, to 6.5 (+/- 1.5) ticks/animal/day on average during the infestation peak, which lasted 6-8 weeks, until early or mid-July. The number then decreased to less than one tick/animal/day from the end of July onwards. The infestation on the predilection sites followed the same trend, with a daily tick burden increase of three to five A. variegatum adults, depending on herd and year, during the infestation peak.     

Borreliacidal activity of saliva of the tick Amblyomma americanum

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.     

A case of Amblyomma testudinarium tick bite in a Korean woman

A case of tick bite was found in the inguinal region of a 74-year-old Korean woman. She was attacked by the tick while working in her vegetable garden in the vicinity of mountain located in Suncheon City, the southern coastal area of the Korean Peninsula. On admission she complained of mild discomfort and itching around the bite area. The causative tick was 23 mm long and had slender pedipalps. The scutum was quite ornate and had eyes at the edge. The genital aperture was located anterior to the level of the coxa II. The spiracular plate was comma-shaped and the anus was surrounded posteriorly by the anal groove. The coxa I had subequal 2 spurs; the external one slightly larger. The spur of coxa IV was slightly longer than those of coxae II and III. The tarsus IV had 2 distinct subapical ventral spurs. It was identified as the fully engorged adult female of Amblyomma testudinarium. This is the first human case of Amblyomma bite in Korea.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.     

Invasion of West African cattle by the tick Amblyomma variegatum

Studies in Cameroon and Burkina Faso examined the invasion process of cattle by adult Amblyomma variegatum Fabricius (Acari: Ixodidae) ticks. Nearly all the ticks picked up in the pasture during grazing were found on the limb ends, near the hooves, where they temporarily attached. Then when the cattle lay down, the ticks moved from the feet towards the predilection sites, where they attached definitively. Many ticks disappeared during this displacement. All the female ticks and approximately 70% of the males were thus unable to attach to the predilection sites as long as the pioneer males had not attached and started to produce attraction-aggregation-attachment pheromones. Nevertheless, A. variegatum females and males attached to the feet in similar numbers during grazing, whether the cattle were already infested or not, indicating that stimuli originating from the hosts are powerful enough to attract both sexes. After attachment of the pioneer males, the number of ticks that successfully reached the predilection sites increased. However, even on infested animals, 40-50% of A. variegatum ticks found near the hooves after the grazing periods disappeared during the night following their capture. When moving from the temporary attachment sites towards the final ones, one-third of the ticks changed the individual host. Considering this two-stage infestation process, it is suggested that a targeted tick control, using a foot-bath, might greatly reduce cattle infestation. In particular, it could be effective in traditional herds, with animals grazing permanently during the day, lying down only once back in the night pen.     

An indirect mechanism by which a protein from the male gonad hastens salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum.

In the adult female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), salivary gland degeneration is triggered by an ecdysteroid, provided the female is above a critical weight (approximately 300-400 mg). In mated females, salivary gland degeneration is virtually complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay can be reversed by the injection of a male reproductive tract homogenate directly into the hemocoel. In this study, we consider a possible mechanism of action for this "male factor." Once mated, male factor likely gains access to its target tissue(s) as a humoral factor. Male factor, however, appears not to act by sensitizing the salivary glands to the action of ecdysteroids. Instead, it appears to act by accelerating the appearance of ecdysteroids in the hemolymph.     

Factors affecting the distributions of the ticks Amblyomma hebraeum and A. variegatum in Zimbabwe: implications of reduced acaricide usage

The ticks Amblyomma hebraeum and A. variegatum are the main vectors of heartwater, a disease of ruminants caused by Cowdria ruminantium, in the agricultural areas of Zimbabwe. At present, A. hebraeum is widely distributed in the dry southern lowveld, and occurs in at least seven foci in the higher rainfall highveld. Amblyomma variegatum occurs in the Zambezi valley and surrounding dry lowveld areas in the northwest. The distribution of A. hebraeum has changed considerably over the past 70 years, while that of A. variegatum appears to have remained fairly static. The distribution patterns of both species in Zimbabwe display anomalous features; the ticks occur in areas of lowest predicted climatic suitability for survival and development and in areas where the densities of cattle, the most important domestic host, are lowest. The only factor favouring the survival of the species in the lowveld habitats in which they occur is the presence of alternative wildlife hosts for the adult stage. Their absence from more climatically favourable highveld habitats appears to have been the result of intensive acaricide treatment of cattle over a long period and a historic absence of significant numbers of wildlife hosts. Eradication of A. hebraeum and A. variegatum by intensive acaricide treatment of cattle can be achieved in the absence of significant numbers of alternative hosts, because of the long attachment and feeding periods of the adults of these tick species. However, eradication becomes impossible when alternative hosts for the adult stage are present, because a pheromone emitted by attached males attracts the unfed nymphal and adult stages to infested hosts. The unfed ticks are not attracted to uninfested hosts, such as acaricide-treated cattle.Regular acaricide treatment of cattle is expensive and so, for economic reasons, the Government of Zimbabwe is no longer enforcing a policy of strict tick control. It is likely that reduced tick control will result in the spread of Amblyomma ticks to previously uninfested areas. Added to this, recent introductions of various wildlife species to highveld commercial farming areas have created conditions in which the ticks could become established in higher rainfall areas. Amblyomma hebraeum is more likely to spread than A. variegatum, because its adults parasitize a wider range of wildlife hosts (warthogs, medium to large-sized antelope, giraffe, buffalo and rhinoceros), whereas adults of A. variegatum appear to be largely restricted to one wildlife species (buffalo) in Zimbabwe, the distribution of which is now confined to very limited areas of the country, as part of foot and mouth disease control measures. A model to predict the rate of spread of A. hebraeum through the highveld is described.Possible control options for dealing with the spread of Amblyomma ticks and heartwater to previous unaffected highveld areas, include (1) continuation of intensive acaricide treatment of cattle to prevent the spread, (2) establishment of a buffer zone of intensive tick control around affected areas to contain the spread and (3) allow the spread to occur and control heartwater by means of immunization. An economic analysis to determine the costs and benefits of the control options, which takes into account the development of Amblyomma-specific tick control technologies and improved heartwater vaccines, is recommended.Deceased.     

Footbath acaricide treatment to control cattle infestation by the tick Amblyomma variegatum

Previous studies have shown that about 90% of adult Amblyomma variegatum Fabricius (Acari: Ixodidae) picked up daily by grazing cattle are still attached to the interdigital areas in the evening, when the animals return from pasture. It was therefore postulated that a targeted treatment, designed to kill the ticks attached to the feet, would limit infestation of the predilection sites. Footbaths filled with various pyrethroid formulations were used over 3 years, at the beginning of the rainy season (from mid-May to the end of July), to assess the efficacy of such a control method. It proved efficient in preventing the ticks from attaching to the predilection sites. Although five to 12 A. variegatum adults attached to each treated animal daily, and although the tick burden of the predilection sites of control cattle increased each day by four to 10 ticks, the average infestation of the predilection sites of treated cattle that were initially highly infested (over 100 ticks/animal) continuously decreased to reach a level of about 10-30 ticks/animal after 6-8 weeks of treatment. In herds with a lower initial tick burden (40-70 ticks/animal) this level was obtained within 2-3 weeks and the mean infestation subsequently remained consistently low. Footbath treatment carried out every other day during the adult peak infestation period should therefore greatly limit losses due to ticks. This method was appreciated by traditional livestock farmers, essentially because it is not time-consuming and because it requires only c. 200 mL aqueous formulation per animal at each passage. The cost of the acaricide needed to treat one animal during the peak infestation period was assessed at c. euro 0.20. This control method might also have an impact on some species of tsetse flies and mosquitoes, thereby contributing to trypanosomiasis and malaria control.     

Infestations of the bont tick Amblyomma hebraeum (Acari: Ixodidae) on different breeds of cattle in Zimbabwe

Infestations of adults and nymphs of Amblyomma hebraeum were counted on Brahman (Br), Brahman x Simmental (BS), Sanga (Sa) and Hereford (He) steers exposed to infested pastures at Mbizi in southern Zimbabwe in 1986–1987. Herefords were always the most heavily infested, while the Sanga tended to carry the fewest ticks with the Brahman and Brahman x Simmental groups being in between. The ratios of the engorged females on the four breeds were 2.3 : 1.4 : 1.4 : 1.0 for He : Br : BS : Sa. The ratios of the standard nymphs were 2.2 : 1.4 : 1.7 : 1.0 for He : Br : BS : Sa. The results confirm earlier observations in Africa and support the view that there are genetic differences between breeds in the expression of resistance to this tick species.Deceased.     

Host location by larvae of the reptile tick Amblyomma vikirri (Acari: Ixodidae)

The newly described tick Amblyomma vikirri has a narrow host range, being found mainly on the Australian lizard, Egernia stokesii and rarely on another lizard, Tiliqua rugosa. Both hosts are in the family Scincidae. Larvae of A. vikirri were as successful in locating E. stokesii as T. rugosa from a range of release distances between 20 and 120 mm from the host. Over this range the proportion of ticks which successfully located hosts declined and the time taken by successful ticks to locate hosts increased with increasing release distance. From 60 mm, larvae of A. vikirri located four other lizard species from the families Scincidae and Agamidae and two non-living targets as successfully as they did E. stokesii. The only evidence that there was any host specificity in the searching behaviour of larvae of A. vikirri was that A. vikirri larvae spent less time paused and questing when they were searching for E. stokesii than when they were searching for T. rugosa. Aponomma hydrosauri, a tick which commonly infests T. rugosa but not E. stokesii, spent less time paused and questing when it was searching for T. rugosa than when it was searching for E. stokesii. However, the results overall suggest that the narrow host range of A. vikirri cannot be explained by any ability of the larvae of that species to discriminate between their natural host and other reptile species.     

Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae)

Eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) inhibited the growth of Escherichia coli and Serratia marcescens (Gram-negative bacteria) in solid culture, but not the growth of Staphylococcus epidermidis, and only marginally the growth of Bacillus subtilis (Gram-positive bacteria). When egg wax was extracted with chloroform/methanol (2:1), the extract contained antibacterial activity, but the denuded eggs did not. When assayed against bacteria in liquid culture, the aqueous phase inhibited the growth of S. epidermidis. However, the activity against E. coli was lost during extraction. The antimicrobial component of the aqueous phase was heat stable (100°C for 10 min), resistant to proteinase K (15 min at 55°C) and to pronase (30 min at 37°C). The antibacterial activity in the aqueous phase increased the permeability of the cell membrane of susceptible bacterial cells within 30 min. However, lysis of the cells was detected by optical density measurements (OD_{600 nm}) only after 1.5 h. The most evident cytological changes observed by transmission electron microscopy were a thickening of the cell wall and the appearance of numerous electron lucent areas within the cytoplasm of treated bacteria. Gené’s organ, the egg-waxing organ in ticks, grew enormously during the first 16 days post-engorgement, and gained antimicrobial activity by day 10 (when oviposition began). This suggests that Gené’s organ is the major source of the antibacterial substance in the egg wax. The vitellogenic hormone in A. hebraeum, 20-hydroxyecdysone, when injected into recently engorged females, did not stimulate growth of Gené’s organ or precocious secretion of antimicrobial activity.     

Ixodid tick species recovered from domestic dogs in Japan

The species of ixodid ticks (Acari: Ixodidae) recovered from domestic dogs in Japan between September to November 2000 and April to June 2001 were identified. A total of 4122 ticks, including 1624 larvae, 1200 nymphs, 1016 females and 282 males were removed from 1221 dogs during these periods. Haemaphysalis longicornis (Neumann) was the most frequently found (40.3% of dogs), followed by H. flava (Neumann) (16.1% of dogs), Rhipicephalus sanguineus (Latreille) (4.8% of dogs) and Ixodes ovatus (Neumann) (4.1% of dogs). Small numbers of H. hystricis (Supino), H. campanulata (Warburton), H. japonica (Warburton), H. ias (Nakamura and Yajima), I. persulcatus (Schulze), I. nipponensis (Kitaoka and Saito) and Amblyomma testudinarium (Koch) were also recovered. In the spring sample, a total of 1408 ticks (78 larvae, 411 nymphs, 792 adult females and 127 adult males) were recovered from 570 dogs. The autumn sample included a larger proportion of larval stage and fewer adult ticks (1546 larvae, 789 nymphs, 224 adult females and 155 adult males). Haemaphysalis longicornis, H. flava and I. ovatus showed a wide geographical distribution from northern to southern Japan, whereas R. sanguineus were mainly distributed in the subtropical Okinawa prefecture with a few exceptions. Dogs in rural areas more frequently carried H. longicornis, H. flava and I. ovatus than dogs in urban or suburban areas, whereas R. sanguineus was more associated with the dogs in urban/suburban areas. Exposure to a garden was significantly associated with R. sanguineus and exposure to woodland was significantly associated with H. flava and I. ovatus. This is the first systematic survey of canine ticks in Japan.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.