Persistent Ehrlichia chaffeensis infection in white-tailed deer.

Four white-tailed deer (Odocoileus virginianus) were inoculated intravenously with a deer-origin isolate (15B-WTD-GA) of Ehrlichia chaffeensis. The course of infection was monitored using indirect fluorescent antibody (IFA), polymerase chain reaction (PCR), and culture over a 9 m period. All deer became rickettsemic within 24 days post inoculation (DPI), and all developed antibody titers >1:64 to E. chaffeensis by 17 DPI. Titers in all deer fell below 1:64 during 87 to 143 DPI. One deer exhibited a second period of seropositivity (peak titer of 1:256) from 207 to 271 DPI but was culture and PCR negative during this period. Rickettsemia was confirmed by reisolation of E. chaffeensis as late as 73 to 108 DPI in three deer. Positive PCR results were obtained from femur bone marrow of one deer and from rumenal lymph node of another (leer at 278 DPI. None of the deer developed clinical signs, hematologic abnormalities, or gross or microscopic lesions attributable to E. chaffeensis. Two uninoculated control deer were negative on all tests through 90 DPI at which time they were removed from the study. Herein we confirm that white-tailed deer become persistently infected with E. chaffeensis, have initial rickettsemias of several weeks duration and may experience recrudescence of rickettsemia, which reaffirm the importance of deer in the epidemiology of E. chaffeensis.

Ticks (Acari: Ixodidae) associated with wild animals in the Pantanal region of Brazil.

This paper describes the identification of ticks from wild animals of the Pantanal region in Brazil as part of a comprehensive study about established and emerging tick-host relationships and related pathological aspects. Eighty-one animals were captured (representing 13 species, six orders), and ticks were found on 63 (78%). Tick species identified included Boophilus microplus (Canestrini), Amblyomma cajennense (F.), Amblyomma parvum Aragão, Amblyomma pseudoconcolor Aragão, Amblyomma scalpturatum Neumann, Amblyomma nodosum Neumann, Amblyomma ovale Koch, and Amblyomma tigrinum Koch. Dragging from grasslands yielded negative results compared with the high concentration of ticks that were collected from leaves in the forests.

Production of pheromones by artificially fed males of the tick Amblyomma maculatum (Acari: Ixodidae).

Aggregation-attachment pheromones are produced only by male ticks of the genus Amblyomma that have imbibed blood for at least 8 d from their mammalian hosts. This report demonstrates that production and release of aggregation-attachment pheromones by Amblyomma maculatum (Koch) males can be induced in vitro by using nonblood media, artificially introduced through capillary tubes, into the male ticks. The presence of these pheromones is demonstrated by using both biological observations on bovine hosts and in vitro experimentation with petri dishes. The attraction of unfed female ticks to the artificially fed males (using the petri dish method) was significantly higher (P < 0.001) than the attraction of unfed females to unfed males. Similarly, attraction and attachment were recorded on bovine hosts. Only 62.8 +/- 17.5% of the females released attached to the bovine host. Of these, 61.9 +/- 19.37% attached around artificially fed males that were placed on the bovine 24 h earlier. This percentage did not differ significantly from the 81.4 +/- 7.1% of the total that attached around males that fed naturally for 8 d. In comparison, only 33.3 +/- 21.9% of the total number of females placed on the host did so after being fed 1 d (control group). The possibility of using capillary feeding as a new tool for investigating the physiology and reproductive behavior of blood-sucking arthropods is discussed.

Ticks associated with wild animals in the Nhecolândia Pantanal, Brazil.

A study of ticks associated with wild animals was carried out from September 1996 to April 1998 at the Fazenda Alegria (21,000 ha), in the Nhecolândia Pantanal, State of Mato Grosso do Sul, Brazil, a sunken plain bordering the upper Paraguay river, located 19 x 08'S; 56 x 46'W. A total of 81 wild animals (13 species, 6 orders) were captured with the aid of nets, and ticks were found on 63 (78%). Tick species identified included Boophilus microplus (Canestrini), Amblyomma cajennense (F.), A. parvum (Aragão), A pseudoconcolor (Aragão), A. scalpturatum (Neumann), A. nodosum (Neumann), A. ovale (Koch), and A. tigrinum (Koch). Dragging from grasslands (campos) yielded negative results compared to the high concentration of ticks, mainly nymphs, that were collected from leaves in the forests (capão). Predominance of immature instars (Amblyomma genera) was observed in the end of winter (August-September). Ticks were associated mainly with coatis, deer (Mazama gouazoubira) and anteater, and these animals may play a role in the epidemiology of tick-transmitted pathogens in the Pantanal if one considers their co-existence with local domestic animals.

The effect of male ticks on the feeding performance of immature stages of Rhipicephalus sanguineus and Amblyomma americanum (Acari: Ixodidae).

A unique group of immunoglobulin-binding proteins (IGBPs), produced by ixodid male ticks during the latter half of their prolonged feeding period, improves the feeding performance of co-feeding females. As a follow-up to this observation, we investigated whether male tick feeding also affects the feeding of other developmental stages. Immature stages of Rhipicephalus sanguineus (Latreeille) and Amblyomma americanum (L.) were fed on rabbits in the presence or absence of conspecific males. The mean weight of larvae and nymphs of both species that fed around males and detached from the host on the first day of dropping was significantly higher than when the immature ticks fed on rabbits in the absence of males. However, larvae of both species and nymphs of R. sanguineus that fed slower and detached on the second day of dropping did not show significant differences in weight. A similar pattern was observed for A. americanum nymphs although, unlike R. sanguineus, the presence of males also influenced the feeding performance of the nymphs that fed slowly and detached on the second day of drop-off. The improved feeding performance demonstrated by immature ticks in the presence of males may be due to immunomodulatory saliva proteins, such as immunoglobulin-binding proteins (IGBPs) that are introduced into the co-feeding site. The results are considered in relation to the distribution of ixodid tick species on their natural hosts.

Comparison of methods for introducing and producing artificial infection of ixodid ticks (Acari: Ixodidae) with Ehrlichia chaffeensis.

Only 29.5 +/- 8.91% of engorged Amblyomma americanum (L.) nymphs that we inoculated with Ehrlichia chaffeensis molted successfully to adults compared with 75.8 +/- 7.46% of engorged nymphs that were not inoculated. However, 65.4 +/- 6.02% of unfed nymphs of this species were exposed for 2 h to E. chaffeensis suspension introduced to them through glass capillaries gained weight. These nymphs were placed on rabbits, and approximately 50% of them completed their feeding and molted successfully to adults. Weight gained was higher (71.8 +/- 17.33% and 69.8 +/- 23.26%) for unfed A. americanum females that fed from capillaries for 2 and 24, h respectively, than for nymphs. Similar values were recorded for Dermacentor variabilis (Say) (61.0 +/- 16.23%) and Rhipicephalus sanguineus (Latreille) (59.0 +/- 18.62%) females after 24 h of capillary feeding. The amount of E. chaffeensis suspension taken in by females of A. americanum, D. variabilis, and R. sanguineus during 24 h of feeding was 11.2 +/- 3.56, 10.9 +/- 4.29 and 6.3 +/- 2.35 microliters, respectively. This volume is equivalent to approximately 12,969, 12,622, and 7,295 infected cells ingested by the species mentioned above. Positive correlation between the volume taken in by the ticks and the weight gained by the females was found, but the initial weight of the unfed females did not effect the weight they gained. The pathogen was found in the females of all 3 species by polymerase chain reaction procedures for at least 7 d, indicating that the capillary feeding method can be successfully used for infecting unfed ticks. The potential use of this method is discussed.

Feeding and respiratory gas exchange in the American dog tick, Dermacentor variabilis.

This study investigated the effect of blood feeding on respiratory gas exchange in the dog tick Dermacentor variabilis. Adult male and female ticks were fed on bovine hosts from 1 to 11days. Females fed slowly for the first 6days and then rapidly engorged on blood 2-3days prior to dropping from the host. Ticks were removed at daily intervals during feeding, weighed and CO(2) emission measured at 25 degrees C using flow-through respirometry. During feeding, females (N=39) showed a 100-fold gain in mass from 5.78+/-1.05mg to 541.15+/-18.60mg while standard metabolic rate (Vdot;co(2)) increased from 0.179+/-0.030&mgr;lh(-1) in unfed ticks to 87.32+/-5.72&mgr;lh(-1) in fully engorged ticks. CO(2) release prior to feeding was highly discontinuous with discrete spiracular bursts of CO(2) emission approximately every 30min. For CO(2) emission measured in detached partially or completely fed ticks, burst frequency became more and more rapid as feeding progressed and changed to continuous sustained CO(2) output during rapid engorgement. In contrast to females, male ticks (N=20) showed little change in mass and maintained discontinuous CO(2) throughout the 11day attachment period on the host. The switch from discontinuous to continuous CO(2) release and presumed increase in respiratory water loss in female ticks is correlated to an increase in metabolic expenditure associated with blood meal digestion rather than any factor relating directly to maintenance of water balance.

The effect of various host species in the feeding performance of immature stages of the tick Hyalomma truncatum (Acari: Ixodidae).

In order to determine the effect of various hosts on the feeding performance of the tick Hyalomma truncatum, were used three mammalian species as hosts. Larvae and nymphs of H. truncatum were fed, under controlled laboratory conditions, on gerbils, guinea-pigs and rabbits. The larvae fed for 4.3 +/- 1.4 days on gerbils, 5.6 +/- 1.3 days on guinea-pigs and 4.7 +/- 1.2 days on rabbits. The mean weights of the larvae which fed in the rabbits, guinea-pigs and gerbils were 0.58 +/- 0.09, 0.46 +/- 0.04 and 0.45 +/- 0.04 mg, respectively. The feeding periods of the nymphs on gerbils, guinea-pigs and rabbits were 7.9 +/- 1.3 and 9.6 +/- 2.2 days respectively. The mean weights of the nymphs which fed on the gerbils, guinea-pigs and rabbits were 22.5 +/- 2.8, 19.7 +/- 1.3 and 15.8 +/- 1.4, respectively. Hyalomma truncatum demonstrated a life cycle of a three-host tick on gerbils and guinea-pigs and of a two-host tick on rabbits. The evolutionary advantage of a two-host cycle over a three-host cycle in metastriate ticks is discussed.

Evidence for attachment pheromones in the Cayenne tick (Acari:Ixodidae).

Evidence is presented for the existence of male-produced attachment pheromones in the tick Amblyomma cajennense (F). Unfed males and females attached significantly faster to shaved sides on bovine hosts where preattached males were present than did adult ticks released in control areas. Attached ticks did not form clusters around the preattached males as described for other species of Amblyomma. They attached in areas under bags where they had been released, but were not necessarily close to the feeding males. Males apparently required at least 4 d of feeding before producing the pheromones. Males that fed for < 3 d failed to induce attachment. A significant increase in attachment of newly introduced males and females was observed only on day 4 after the feeding of the preattached male started. This response increased on days 5 and 6 after feeding had commenced. An evolutionary trend in the production of tick pheromones was suggested.

Survival of six species of African ticks in relation to saturation deficits.

The survival of unfed males and females of six species of African ticks was monitored at five different saturation deficits at constant temperature (25 degrees C). The survivorship curves for each species comprised a pre-mortality period, prior to when ticks started to die and a mortality period corresponding to a rapid increase in the mortality rate. Longevity was defined as pre-mortality plus mortality. A negative correlation between the longevity of the ticks and the saturation deficits was found with ticks surviving longer at lower deficits. The survival of males and females was similar. At low saturation deficits (2-4 mmHg) Amblyomma hebraeum survived the longest periods (74 weeks). Some correlation was found between the tick survival under dehydrating conditions and habitat associations. Rhipicephalus appendiculatus and Haemaphysalis leachii, the most mesic in distribution, had the shortest longevity (21 and 13 weeks, respectively) at high saturation deficits (7-21 mmHg). Hyalomma marginatum rufipes, the most xerophilic in distribution, had the longest survival (39.3 +/- 10.5 weeks) at high saturation deficits. Other factors apart from the adult survival should be taken into account when accounting for the tick distribution, in particular the tolerance of earlier developmental stages to desiccation.

The effect of host resistance on the metabolic rate of engorged females of Rhipicephalus evertsi evertsi.

This study investigated the effect of acquired resistance in guinea-pigs on the metabolic rate of adult females of the tick Rhipicephalus evertsi evertsi. Guinea-pigs were subjected to three successive infestations of ticks and the rate of CO2 production (VCO2) measured in first and third infestation engorged females. Ticks which fed on resistant hosts showed a 52% decrease in mass compared to ticks that fed on naive animals. Reduction in mass was accompanied by a decrease in VCO2 (mlh-1) per tick but an increase in mass specific VCO2 (mlg-1h-1). However, both groups shared a single allometric relationship between body mass and metabolic rate (VCO2). We suggest that the differences in size rather than any factor directly relating to the mechanism of acquired resistance account for the differences in metabolic rate between ticks fed on naive and resistant guinea-pigs.

Seasonal abundance of the tortoise tick Amblyomma marmoreum (Acari: Ixodidae) on the leopard tortoise, Geochelone paradalis.

A survey of Amblyomma marmoreum Koch ticks recorded from the leopard tortoise, Geochelone paradalis Bell, in the National Zoological Gardens, Pretoria, South Africa, was carried out over a 19-mo period. No significant differences were found between the tick burdens on male and female tortoises. A. marmoreum showed a clear seasonal pattern of abundance. Larvae were present on the tortoise host during late summer, with a peak in February and March; nymphs were abundant during winter, with a peak in June and July. Male ticks were found throughout the year, but females were present only during spring and early to midsummer with a peak in October. It was demonstrated that each developmental stage of A. marmoreum showed only one peak of activity per year.

The effects of tick numbers and intervals between infestations on the resistance acquired by guinea-pigs to adults of Rhipicephalus evertsi evertsi (Acari: Ixodidae).

Guinea-pigs were infested three times at short (1 week) intervals or long (10 week) intervals between successive infestations with low (1), medium (8) and high (15) numbers of adult Rhipicephalus evertsi evertsi. To compare the resistance developed by the hosts at short and long intervals between infestations, a challenge (fourth) infestation was performed with the high number of ticks. Resistance was assessed by comparing the mean weight of engorged female ticks that dropped from the hosts. Concentrations of beta globulins in the host sera were also monitored. Different tick loads (low, medium and high) and infestation intervals had a similar influence on the reduction in weight of the females which was between 60-70% after the final challenge. However, the concentration of beta globulins, was highest in the guinea-pigs exposed to medium and high numbers of ticks with long infestation intervals, probably because of an increase in the production of protective antibodies in response to more intensive antigenic challenge. The relationship between engorgement weight and beta globulin levels in host sera is discussed.

Isotopes as host blood markers to measure blood intake by feeding ticks (Acari: Ixodidae).

We have modified an experimental technique in which two different isotopes (Cr51 and I125) were used simultaneously as blood markers for determining the amount of blood removed by feeding ticks. This method enables us to measure separately and directly the volumes of red blood cells and plasma in ticks and to calculate the ratio between the two parameters (concentrating ability). The concentration of red blood cells in nymphs of Amblyomma hebraeum Kock and Rhipicephalus appendiculatus Neumann and in females of these species plus in those of Hyalomma truncatum Kock were found to be twice the amount measured in the labeled blood of goats. Concentrations of plasma in the ticks were approximately two-thirds of that measured in samples taken directly from these same goats. Small nonsignificant differences between the ability of various tick species to concentrate red blood cells and dilute host plasma were found. The advantage of this method is compared with that of the colorimetric and gravimetric methods, and its potential use is discussed.

Respiratory gas exchange in the tick Amblyomma hebraeum (Acari: Ixodidae).

Respiratory gas exchange was studied in unfed adult Amblyomma hebraeum Koch. Carbon dioxide emission was measured at 25 degrees C using flow-through respirometry to determine standard metabolic rate and the temporal pattern of gaseous emission. The standard CO2 production rate (sVCO2) of inactive ticks was 0.0135 +/- 0.0085 ml g-1 h-1, and the standard O2 consumption rate (sVO2) was 0.0158 +/- 0.0097 ml g-1 h-1. Ventilation was discontinuous and was characterized by periodic bursts of CO2 emissions at frequencies of 0.33 h-1-1.11 h-1. Low metabolic rate coupled with discontinuous ventilation may contribute to the ability of adult A. hebraeum to withstand prolonged periods of starvation and desiccation in the absence of a host.

Oxygen consumption of engorged nymphs and unfed adults of Hyalomma asiaticum ticks (Acari: Ixodidae) exposed to various photoperiods.

The oxygen consumption of engorged nymphs of Hyalomma asiaticum was measured at various intervals after drop-off from mice hosts. Duration of nymphal development to the emergence of adults was 25-32 days at 25 degrees C. The oxygen consumption was high immediately after completing the blood meal (193-248 mm3 g-1 h-1 but decreased significantly 18 days later (at 25 degrees C) to 45-65 mm3 g-1 h-1. It increased again before ecdysis (81-102 mm3 g-1 h-1, and also after ecdysis in freshly moulted adults (177-220 mm3 g-1 h-1. The oxygen consumption in 8-month-old adult ticks was very variable ranging from 40-42 to 172 mm3 g-1 h-1. Neither engorged nymphs nor unfed adult ticks showed any dependence of their respiratory metabolism on the photoperiodic regimes tested (LD 20:4 and LD 12:12, with or without transfer to an alternative photoperiod after engorgement of nymphs).

Selected biochemical parameters in captive mountain tortoises (Geochelone pardalis).

The concentrations of sodium, potassium, chloride, urea, creatinine, uric acid, total protein, albumin, inorganic phosphorous, calcium, magnesium, cholesterol, triglyceride, glucose, aspartate and alanine transaminases (AST and ALT), creatine kinase (CK), lactic acid dehydrogenase (LD), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP) and total bilirubin in the serum of a captive population of the mountain tortoise (Geochelone pardalis) (n = 13) were determined. Results varied considerably, particularly for most enzymes such as AST (11-113 U/l,ALT (1-72 U/l), CK (12-242 U/l), LD (147-2641 U/l) and ALP (56-168 U/l).

Ventilation in the adults of Amblyomma hebraeum and A. marmoreum (Acarina, Ixodidae), vectors of heartwater in southern Africa.

The objective of this study was to establish the major features of respiratory gas exchange in unfed adults of the ticks Amblyomma hebraeum and A. marmoreum, both vectors of heartwater in Southern Africa. Carbon dioxide emission of ticks was measured at 25 degrees C using flow-through respirometry in order to determine standard metabolic rate (SMR) and the temporal pattern of gaseous emission. For both species, SMR was extremely low and approximately 100 fold less than that predicted for an insect of equivalent body mass. Ventilation in inactive ticks was discontinuous and characterized by periodic bursts of CO2 emissions during spiracular opening. The main selective advantage of this type of ventilation is believed to lie in a reduction of respiratory water loss. The periodicity of CO2 bursts was less frequent in A. marmoreum (every 2.5 h) compared to A. hebraeum (every 1.5 h) suggesting that A. marmoreum is more efficient at conserving respiratory water loss. It is suggested that future research into water balance physiology of ticks should address the role of ventilatory patterns in determining off-host survival and habitat associations.

The effects of nutritional status of rabbits and sheep on their resistance to the ticks Rhipicephalus evertsi evertsi and R. appendiculatus.

Rabbits and sheep were exposed to low- and high-protein diets and subsequently infested three times with adults of Rhipicephalus appendiculatus and Rhipicephalus evertsi evertsi. The mean weight of R.e. evertsi females which dropped from rabbits maintained on a high-protein diet decreased from 515.0 +/- 24.9 mg (naive) to 381.5 +/- 25.0 (second infestation) to 340.3 +/- 23.3 mg (third infestation) while the weight of ticks fed on animals which were exposed to a low-protein diet did not change significantly (2.7%). The mean weight of engorged females of R. appendiculatus which completed their blood meal on rabbits (high protein) decreased from 520.9 +/- 31.8 (naive) to 369.3 +/- 39 mg (3rd infestation), a significant decrease of 29.1% compared to a 12.3% decrease in weight between the 1st and 3rd infestation of females fed on animals on a low-protein diet. Rhipicephalus e. evertsi fed on sheep exhibited the same phenomenon. The mean decrease in weight of 4rd-infestation ticks which dropped from sheep fed lucerne was 26.2% compared to 16.6% for ticks from sheep which were fed on grass. Hosts maintained on a low-protein diet failed to acquire resistance to ticks, lost weight and developed anaemia while those on a high-protein diet developed resistance, maintained weight and did not develop anaemia. The nutritional stress of the hosts and its application in South Africa are discussed.

Laboratory studies on the life cycle of Amblyomma marmoreum (Acari: Ixodidae) on two different hosts.

A comparative study was made of the life cycle of the tortoise tick, Amblyomma marmoreum Koch, on tortoises and guinea pigs under laboratory conditions. At 25 degrees C and 85% RH with natural day length, duration of off-host stages (preoviposition, oviposition, incubation, and premolt) was similar for ticks fed on both hosts. Delay in preoviposition (up to 90 d) was observed in some gravid females. Larvae, nymphs, and adults had longer feeding periods on tortoises than on guinea pigs. Adult females fed on tortoises had greater engorgement wieght and ovipositional capacity than ticks fed on guinea pigs. It is suggested that the shortened feeding period of immature stages on mammalian hosts together with the occurrence of morphogenic diapause may be more important than previously recognized in determining whether the life cycle of A. marmoreum is completed in 1 or 2 yr under natural conditions.