Co-feeding studies of ticks infected with Anaplasma marginale

Ticks often cluster at preferred feeding sites on hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. While the major route of infection of ticks with pathogens is via the bloodmeal during feeding on a parasitemic host, non-systemic transmission of viruses and spirochetes has been shown to occur from infected to uninfected ticks at common feeding sites on uninfected hosts. In this research, two separate studies were done using the tick-borne rickettsial pathogen of cattle, Anaplasma marginale. In one study we tested whether A. marginale could be transmitted non-systemically from infected to uninfected Dermacentor variabilis males while co-feeding on rabbits. Infection of ticks was determined by allowing them to transmission feed on susceptible cattle and by DNA probe and microscopy studies on salivary glands. In the second study, we tested whether the co-feeding of male and female ticks on parasitemic cattle would increase the acquisition and development of A. marginale in males. A. marginale infections in salivary glands were determined by quantitative PCR after the ticks were allowed to transmission feed on susceptible cattle. Non-systemic transmission of A. marginale did not occur from infected and uninfected ticks that fed at the same site on rabbits and, therefore, does not appear to be a means of A. marginale transmission. A. marginale infections in male ticks were not increased while co-feeding with females. Thus, co-feeding of adult Dermacentor spp. does not appear to influence the dynamics of A. marginale transmission.     

Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever

Q fever has emerged as an important human and veterinary public health problem in the Netherlands with major outbreaks in three consecutive years. Goat farms are probably the prime source from which Coxiella burnetii have spread throughout the environment, infecting people living in the vicinity. Coxiella burnetii infection not only spilled over from animal husbandry to humans but could also have spread to neighbouring wildlife and pets forming novel reservoirs and consequently posing another and lingering threat to humans, companion animals and livestock. In these cases, transmission routes other than airborne spread of contaminated aerosols may become significant. Therefore, the role of ticks in the transmission of Coxiella burnetii in the current situation was investigated. A total of 1891 questing Ixodes ricinus ticks and 1086 ticks feeding on pets, wildlife and livestock were tested by a recently developed multiplex Q-PCR. All ticks were negative, except for a few ticks feeding on a herd of recently vaccinated sheep. Coxiella-positive ticks were not detected after resampling this particular herd three months later. Based on these data we conclude that the current risk of acquiring Q fever from questing ticks in the Netherlands is negligible. However, for future risk assessments, it might be relevant to sample more ticks in the vicinity of previously C. burnetii infected goat farms and to assess whether C. burnetii can be transmitted transovarially and transstadially in I. ricinus ticks.     

Distribution and biology of Ornithodoros erraticus in parts of Spain affected by African swine fever

Ornithodoros erraticus was found in 30.7 per cent, 35.0 per cent and 71.0 per cent of the pig-pens sampled in the provinces of Salamanca, Badajoz and Huelva in which African swine fever is a problem in the rearing of Iberian pigs. Between 38 and 65 per cent of the pig-pens in these areas are now abandoned and their populations of O erraticus are extinct or becoming so because they can no longer feed on pigs, which in Spain are their main hosts. The abandonment of pig-pens has resulted in the elimination of most soft ticks infected with the virus of African swine fever, and means that the distribution of ticks is now irregular and focal. Another factor affecting their distribution is the kind of soil on which the pig-pens are located. In abandoned pig-pens, the adults and large nymphs survive for about five years or longer when animals occasionally enter them. Hungry tick populations may transmit African swine fever when feeding in winter, whereas the populations that have continuous access to pigs do not feed until the pig-pens reach a temperature of 13 to 15 degrees C. In the latter populations, each stage exhibits a single annual peak of activity, which implies that the development from larva to adult takes two to three years. Pigs may die as a result of the bites, but on no occasion were 100 per cent of the fasting ticks seen to feed, even though they had the opportunity of doing so. This may hinder the eradication of this soft tick from infested pig-pens.     

Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24-27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats.     

The transovarial transmission of Babesia caballi by Hyalomma truncatum

Babesia caballi, isolated from a horse that originated from South West Africa/Namibia, was transmitted transovarially by adult Hyalomma truncatum. B. caballi proved to be highly infective for adult H. truncatum. Forty-five per cent of ticks feeding on a reacting animal with an extremely low parasitaemia became infected. In spite of a low parasitaemia, the ticks were severely affected by the parasite. Seventy per cent of the infected ticks either died during oviposition or after laying only a few eggs. The features of the infection in horses were: a prepatent period of 10 days, very low parasitaemias with low pathogenicity and spontaneous recovery of the infected animals.     

Evidence for regurgitation by Amblyomma americanum

Western blot analysis of Amblyomma americanum-derived midgut extracts using tick-resistant rabbit and guinea pig antisera demonstrated several unique polypeptides ranging from 7 to 355 kDa. These polypeptides were not observed in similar blots of salivary gland extracts or serum from the respective host animals. The data from this study suggest that female A. americanum ticks regurgitate gut material during feeding. Such a finding explains the ability of immunization with gut-derived preparations to induce immunity and may help to understand the mechanism of transmission of several tick-borne pathogens that have been only clearly demonstrated to infect the gut of ticks.     

Experimental infection by capillary tube feeding of Rhipicephalus sanguineus with Bartonella vinsonii subspecies berkhoffii

It has been speculated that ticks may serve as vectors of Bartonella species. Circumstantial, clinical, epidemiological and serological evidence suggest that B. vinsonii subspecies berkhoffii (B. v. berkhoffii) might be transmitted by Rhipicephalus sanguineus. The purpose of the present study was to determine whether adult R. sanguineus ticks can be infected with a B. v. berkhoffii genotype II isolate via capillary tube feeding and whether the infection can then be transmitted from adult females to their eggs via trans-ovarial transmission. Furthermore, tick fecal material was also collected and screened as a possible source of infectious inoculum for canine infections. B. v. berkhoffii DNA was detected in 50% (7 of 14) of females that did not oviposit and in 14.3% (2 of 14) of female ticks that laid eggs, but not detected in egg clutches (100 eggs/female). DNA was also detected in tick feces collected on days 2 through 6 post-capillary tube feeding, however, dogs (n=3) did not become bacteremic or seroconvert when inoculated with tick fecal material. Therefore, trans-ovarial transmission of B. v. berkhoffii by R. sanguineus is unlikely, but further studies are needed to determine if tick fecal material can serve as a source of infection to canines.     

Intermediate site of development of Anaplasma marginale in feeding adult Dermacentor andersoni ticks that were infected as nymphs

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.     

Morphology and development of Cowdria ruminantium in Amblyomma ticks

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.     

Movement of Rhipicephalus sanguineus adults between co-housed dogs during active feeding

Adult male ticks have been shown capable of experimental acquisition and transmission of tick-borne pathogens without requiring a molt. To determine the ecological relevance of this intrastadial transmission route, we evaluated the extent to which actively feeding male Rhipicephalus sanguineus move naturally between co-housed infested dogs. Dogs (n=4) were infested with single color-coded ticks, individually housed in tick-confinement cages for 48 h while infestations established, and then each dog examined and the ticks present counted. Dogs were then co-housed in a large, group pen for an additional 7 (study 1) or 5 (study 2) days. In the first study, sex ratios were adjusted to encourage migration, with two dogs receiving predominantly female ticks and two dogs receiving all male ticks. In the second study, each dog received a ratio of ticks that parallels that found in natural infestations (4:1 male to female). Results showed that ticks readily migrated between infested, co-housed dogs. Rates of immigration, defined as the percentage of ticks previously attached to one dog that moved onto another dog, ranged from 0 to 46% (mean=31.1% study 1; 9.4% study 2). Emigration rates, defined as the number of ticks initially infesting one dog that moved to another dog, averaged 35.2% in study 1 and 10.8% in study 2 (3.6-67.6%). Movement of adult ticks between dogs represents a naturally occurring form of interrupted feeding, a strategy which has been shown to shorten the feeding time necessary to allow transmission of pathogens. In ticks that readily detach from one host and reattach to a second host to resume feeding, replication of any pathogens present has already been initiated and therefore the same delay in transmission seen in ticks attached to a host for the first time may not occur.     

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.     

Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.     

Rearing and infection techniques for Amblyomma species to be used in heartwater transmission experiments

Techniques for the rearing of Amblyomma species to be used in heartwater (Cowdria ruminantium) transmission experiments are discussed. These involve the breeding and maintenance of infected and non-infected strains of ticks. They include the feeding of ticks on sheep, rabbits, mice, tortoises and guinea fowl.     

Heartwater. The artificial transmission of Cowdria ruminantium in domestic ruminants and mice

The artificial transmission of Cowdria ruminantium with infected blood, organ homogenates, peritoneal macrophages, tick stabilate and tissue culture cells is discussed. Organ homogenates prepared from the myocardium, spleen, kidneys and liver of diseased animals are commonly used to infect mice. The efficacy of organ homogenates as a source of C. ruminantium depends on factors such as the route of inoculation and the heartwater isolate used. Heartwater is artificially transmitted with infected tick stabilate, haemocytes, rectal ampules and hypodermal homogenates. The infectivity of saliva collected from Amblyomma hebraeum female ticks was very low compared to the ground-up suspensions prepared from the same group of ticks.     

Transmission of Anaplasma marginale by adult Dermacentor andersoni during feeding on calves

Laboratory-reared Dermacentor andersoni ticks experimentally infected as nymphs with Anaplasma marginale were allowed to feed as adults from 1 to 9 days on susceptible, splenectomized calves to determine when, during feeding, the hematozoan was transmitted from ticks to cattle. In experiment 1, ticks were allowed to feed on calves for 1, 2, 3, 4, 5, or 6 days and anaplasmosis did not result. The same calves were used for experiment 2, and ticks were allowed to feed for 1, 3, 6, 7, 8, or 9 days and anaplasmosis occurred in all calves on which ticks fed for greater than or equal to 6 days. In 2 trials in experiment 3, ticks were allowed to feed on calves for 1 to 9 days. Anaplasmosis developed only in calves on which ticks fed for 7, 8, or 9 days. The prepatent periods shortened with longer tick feeding, and linear regression analysis of combined prepatent periods of both trials of experiment 3 indicated a significant (P = 0.05) slope with an estimated daily decrease of 7.75 days from day 7 to 9 of feeding. There was no apparent correlation between length of tick feeding and severity of clinical signs in those calves that developed anaplasmosis. Seemingly, A marginale can be transmitted to cattle by adult D andersoni ticks no earlier than the 6th or 7th day of feeding.     

Infectivity of three Anaplasma marginale isolates for Dermacentor andersoni

Three isolates of Anaplasma marginale--Virginia (VAM), Illinois (IAM), and Florida (FAM)--were compared for infectivity for Dermacentor andersoni. The isolates were selected, in part, because of a tail-like appendage that has been demonstrated in the VAM and IAM, but not in the FAM. Ticks were exposed to the isolates as nymphs either naturally by feeding on a calf with anaplasmosis or artificially by percutaneous inoculation with infected bovine erythrocytes. They were examined for infectivity after molting to the adult stage by determining their capability to transmit the disease to susceptible calves and by demonstrating colonies in tick gut sections. Only those ticks exposed to the VAM proved to be infected with A marginale; ticks naturally exposed and those artificially infected with this isolate transmitted the disease to susceptible calves. Colonies of A marginale were observed only in gut tissues of ticks naturally infected with VAM. The IAM (appendage present) and FAM (appendage absent) could not be found in ticks exposed by either method, indicating that factors other than the presence of inclusion appendages may be involved in infection of ticks by A marginale.     

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

Experimental transmission of a granulocytic form of the tribe Ehrlichieae by Dermacentor variabilis and Amblyomma americanum to dogs

Transstadial transmission of granulocytic Ehrlichieae in dogs was attempted using ticks, Amblyomma americanum and Dermacentor variabilis. Ticks were exposed by feeding as nymphs on acutely infected pups; adult ticks then fed to repletion on susceptible adult dogs that were monitored daily for signs of infection. Evidence of transmission was not observed in control dogs or in those exposed to D variabilis. In contrast, dogs exposed to A americanum developed serologic or clinical evidence of infection, but organisms were not seen in blood smears until corticosteroids were administered, causing recrudescence and accompanying parasitemia. At 12 days after subinoculation of blood obtained from donor adult dogs, before corticosteroids were administered, a febrile response, thrombocytopenia, and appearance of morulae in neutrophilic granulocytes were observed in 2 susceptible recipient dogs.