Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.     

Distribution and molecular characterization of Wolbachia endosymbionts and filarial nematodes in Maryland populations of the lone star tick (Amblyomma americanum)

The lone star tick Amblyomma americanum is host to a wide diversity of endosymbiotic bacteria. We identified a novel Wolbachia symbiont infecting A. americanum. Multilocus sequence typing phylogenetically placed the endosymbiont in the increasingly diverse F supergroup. We assayed a total of 1031 ticks (119 females, 78 males and 834 nymphs in 89 pools) from 16 Maryland populations for infection. Infection frequencies in the natural populations were approximately 5% in females and <2% (minimum infection rate) in nymphs; infection was not detected in males. Infected populations were only observed in southern Maryland, suggesting the possibility that Wolbachia is currently invading Maryland A. americanum populations. Because F supergroup Wolbachia have been detected previously in filarial nematodes, tick samples were assayed for nematodes by PCR. Filarial nematodes were detected in 70% and 9% of Wolbachia-positive and Wolbachia-negative tick samples, respectively. While nematodes were more common in Wolbachia-positive tick samples, the lack of a strict infection concordance (Wolbachia-positive, nematode-negative and Wolbachia-negative, nematode-positive ticks) suggests that Wolbachia prevalence in ticks is not due to nematode infection. Supporting this hypothesis, phylogenetic analysis indicated that the nematodes were likely a novel species within the genus Acanthocheilonema, which has been previously shown to be Wolbachia-free.     

Persistence of Hepatozoon americanum (Apicomplexa: Adeleorina) in a naturally infected dog

To determine the persistence of Hepatozoon americanum in a naturally infected dog, skeletal muscle biopsies were performed at approximately 6-mo intervals over a period of 5.5 yr, and the samples were examined for presence of lesions of American canine hepatozoonosis (ACH). Nymphal Amblyomma maculatum (Gulf Coast tick) were allowed to feed to repletion on the dog periodically over the 5.5-yr period, and adult ticks were dissected and examined for presence of H. americanum oocysts. With 3 exceptions, the biopsied muscle contained lesions characteristic of ACH; no evidence of infection was found at 36, 54, and 67 mo after the original diagnosis. In every instance, nymphal Gulf Coast ticks became infected, indicating that dogs naturally infected with H. americanum can remain infectious for Gulf Coast ticks for at least 5.5 yr. Skeletal muscle biopsy is a reasonably reliable method of determining whether dogs are infected with the parasite. Xenodiagnosis using nymphal Gulf Coast ticks is an even more sensitive method, but the procedure is practicable only experimentally. Design of prevention and control measures for ACH must take into account knowledge that the parasite can survive in dogs, and presumably other vertebrate host(s), for long periods. Preventing ingestion of Gulf Coast ticks is an effective control measure.     

Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.     

Electron-Microscopic Studies on Theileria ovis Rodhain, 1916: Development of Kinetes in the Gut of the Vector Tick, Rhipicephalus evertsi evertsi Neumann, 1897, and Their Transformation Within Cells of the Salivary Glands

SYNOPSIS. Gamogony of Theileria ovis Rodhain occurs within the gut of nymphs of the tick Rhipicephalus evertsi evertsi Neumann. After molting, spherical and ovoid parasites are found within the intestinal cells of the ticks. These stages are thought to be zygotes, because they undergo a transformation process leading ultimately (in 3 days) to the formation of a motile stage, the kinete , the fine structure of which is very similar to that of the ookinetes of the hemosporidia. The kinete leaves the gut cells of the tick and penetrates the salivary gland cells where it produces infective stages (the sporozoites ). These stages may be transmitted to sheep during the next blood meal of the tick. The developmental processes of T. ovis are compared to those of Hemosporina.     

Electron-microscopic studies on Theileria ovis Rodhain, 1916: development of kinetics in the gut of the vector tick, Rhipicephalus evertsi evertsi Neumann, 1897, and their transformation within cells of the salivary glands.

Gamogony of Theileria ovis Rodhain occurs within the gut of nymphs of the tick Rhipicephalus evertsi evertsi Neumann. After molting, spherical and ovoid parasites are found within the intestinal cells of the ticks. These stages are thought to be zygotes, because they undergo a transformation process leading ultimately (in 3 days) to the formation of a motile stage, the kinete, the fine structure of which is very similar to that of the ookinetes of the hemosporidia. The kinete leaves the gut cells of the tick and penetrates the salivary gland cells where it produces infective stages (the sporozoites). These stages may be transmitted to sheep during the next blood meal of the tick. The developmental processes of T. ovis are compared to those of Hemosporina.     

Experimental infection of adult and juvenile coyotes with domestic dog and wild coyote isolates of Hepatozoon americanum (Apicomplexa: Adeleorina)

Each of five adult and four juvenile coyotes (Canis latrans) was exposed to an oral dose of 50 Hepatozoon americanum oocysts recovered from Amblyomma maculatum ticks that previously fed on either naturally infected domestic dogs (Canis familiaris) or naturally infected wild coyotes. All coyotes exposed to H. americanum became infected, regardless of isolate source, and all exhibited mild to moderate clinical disease that simulated American canine hepatozoonosis in naturally infected dogs. At 100 days postexposure, parasitemia was greater in juvenile than adult coyotes (0.9% and 0.3%, respectively); radiographic imaging of femurs revealed moderate exostosis in all juveniles and mild to moderate new bone growth in four of five (80%) adult coyotes. Gross postmortem analysis of bone lesions demonstrated variation between age groups of coyotes but not between isolates of H. americanum. Microscopic evaluation of skeletal muscle revealed that parasite-induced lesions were significantly more numerous (t = 5.0, df = 7, P = 0.001) in juvenile than adult coyotes. Results of this study indicate that juvenile and adult coyotes are equally susceptible to experimental infection with H. americanum isolated from domestic dog and wild coyote sources. The age of coyotes at the time of exposure, and possibly the number of H. americanum oocysts ingested, might influence morbidity and mortality, but it appears that both adult and juvenile coyotes could be reservoirs of H. americanum.     

Morphology and structural organization of Haller's organ during postembryonic development ofArgas (Persicargas) walkerae (Ixodoidea: Argasidae)

Scanning-electron-microscopic investigations of Haller's organ in larvae, nymphs I, II, III and IV, and male and female adultArgas (Persicargas) walkerae ticks showed that morphology and structural organization change during postembryonic development. Stage-dependent differences existed regarding setal numbers of the anterior pit as well as formation and reticulation of cuticular projections in the capsule cavity. The anterior pit increased in size in the course of postembryonic development. It contained only seven setae in larvae, one conical, setiform and grooved seta each as well as two porose and fine setae. Nymphs I, II, III and IV and adult ticks had equal numbers of setae; however, one additional unilaterally serrate and grooved seta each were present. Setal length increased continuously during postembryonic development and attained maximum values in adult ticks. The capsule consisted of roof and cavity and was located distinctly lateral in larvae, slightly lateral in nymphs I and II, and in all other stages directly on the longitudinal axis of tarsus. The capsule roof showed a reticular structure. The slit-like main aperture was located peripherally and arranged transversally to the longitudinal axis of tarsus I in larvae. Nymphs and adult ticks had a central, circular main aperture. Stage-dependent cuticular projections of varying form protruded into the capsule cavity. Larvae had only single, free-standing projections which ramified slightly and communicated with each other. Projections were more heavily reticulated in nymphs I and II. In nymphs III and IV as well as male and female adult ticks, a long centrally situated tube of reticular appearance was seen, which was supported by a large number of radially organized and interlocking pillars and communicated with the capsule roof. In all tick stages there were always four porose setae present, arranged on the capsule floor.     

Borrelia sp. in ticks recovered from white-tailed deer in Alabama.

Six hundred sixty-five hunter-killed white-tailed deer (Odocoileus virginianus) from 18 counties in Alabama (USA) were examined for ticks. Most of the collections were made at state-operated wildlife management areas. Four species of ticks (n = 4,527) were recovered: the lone star tick Amblyomma americanum (n = 482); the Gulf Coast tick A. maculatum (n = 11); the winter tick Dermacentor albipictus (n = 1,242); and the black-legged tick Ixodes scapularis (n = 2,792). Fifty-six percent of the ticks (n = 2,555) were examined for Borrelia sp. spirochetes using an immunofluorescent, polyclonal antibody assay. Spirochetes were detected in I. scapularis (five females, seven males) from Barbour, Butler, Coosa, and Lee counties and A. americanum (four males, four nymphs) from Hale, Lee, and Wilcox counties. Area-specific prevalences in ticks were as high as 3.3% for I. scapularis and 3.8% for A. americanum.     

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.     

The longevity of the tick Ixodes ricinus (Acari: Ixodidae) in Central Russia

The longevity of the tick Ixodes ricinus has been studied on 27 samples from a wild population of the Moscow Region. Ticks were kept in small cages and glass tubes under conditions close to natural. About 2/3 of the specimens collected in spring survived till the next spring and later died out gradually during the second season of their activity. Very few specimens reached the beginning of the third winter in their life, but apparently none of them survived the end of the winter. The life span of males was the same as that of females. No mass activation of adult ticks was recorded in the autumn of the year of their molting. It seems that the rise of the autumnal density of adult ticks depends mainly on the increased activity of the females which molted from nymphs last year.     

The seabird tick, <Emphasis Type="Italic">Ixodes</Emphasis><Emphasis Type="Italic">uriae</Emphasis>, uses uric acid in penguin guano as a kairomone and guanine in tick feces as an assembly pheromone on the Antarctic Peninsula

In the vicinity of Palmer Station, Antarctica, the seabird tick, Ixodes uriae, forms large aggregations under rocks at the periphery of Adelie penguin rookeries. When the adult penguins return to the rookeries at the beginning of the nesting season the ticks leave their off-host aggregation site, attach to the penguins for a period of feeding, and then subsequently return to the aggregation site. In this study, we searched for chemical cues that may be used by the ticks to locate their aggregation sites as well as cues involved in finding penguins. Tick excreta and soil extracts from beneath tick aggregations contained a pheromone that elicited assembly behavior in unfed larvae, non-fed nymphs and non-fed adults. Guanine, the major excretory product of ticks, elicited assembly behavior, thus, guanine is likely an active component involved in assembly. Non-fed stages also responded positively to penguin guano and uric acid, the primary excretory product of penguins, suggesting that uric acid and other components of penguin guano function as a kairomone used by the non-fed ticks to locate their host. After feeding, the immature ticks’ response to both the assembly and kairomones is switched off for several days, and the ticks regain responsiveness only after they have molted. Fed adult females lay eggs and die without ever regaining responsiveness. Thus, I. uriae relies on two closely related chemicals to regulate two critical aspects of its life: assembly and host-finding. Guanine and other components of tick excreta function as an assembly pheromone in promoting the formation of off-host aggregations, while uric acid and other components of penguin guano function as a kairomone used by the tick to locate its host.     

Observations on Antricola ticks: small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccessful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gut.     

Biology of Francisella tularensis subspecies holarctica live vaccine strain in the tick vector Dermacentor variabilis

Background

The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis.

Methodology/Principal Findings

Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8±0.8×10¹ and 1.1±0.03×10³ CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae.

Conclusions/Significance

This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the central United States.     

Observations on the Development of Babesia caballi (Nuttall) in the Tropical Horse Tick Dermacentor nitens Neumann

SYNOPSIS. The development of Babesia caballi (Nuttall) in Dermacentor nitens Neumann was studied in smear preparations and histologic sections of ticks infected with this protozoan parasite. A majority of the parasites in equine erythrocytes ingested by the adult ticks apparently were destroyed. Smal spherical bodies 4–6 μ in diameter were the 1st developmental stages of B. caballi observed in the gut contents of ticks infected with this parasite. These spherical bodies apparently gave rise to clavate (club-shaped) bodies 10–14 μ long by 4–6 μ wide. The latter developed into large round bodies 12–16 μ in diameter that segmented into vermicular-shaped parasites, about 8–12 μ long by 2–4 μ wide; some penetrated the gut wall, some invaded other cells of the tick.
In the cells of the Malpighian tubules, hemolymph, and ovaries, the vermicular parasites underwent a secondary cycle of multiple fission, forming vermicules similar to those occurring earlier in the gut. Vermicules that invaded the ova underwent a similar multiple fission cycle during the larval stage of the tick.
Vermicules from the multiple fission cycle that occurred during the period of larval feeding invaded the salivary glands. A multiple fission cycle of increase within these glands resulted in large numbers of small, oval and piriform parasites, 2.5–3 μ, maximum dimension. These parasites became mixed with the salivary secretions, and presumably are the forms injected into the horse by the nymphs as they feed. The small oval and piriform parasites therefore appear to be the infective stage for the horse.     

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

Ticks (Acari: Ixodidae) infesting the Arabian Camel (Camelus dromedarius) in the Sinai,Egypt with a note on the acaricidal efficacy of Ivermectin

In this study, tick burdens on camels (Camelus dromedarius) were determined in the vicinity of the St. Catherine monastery, Sinai, Egypt. In total 2,545 ticks (1,491 adults and 1,054 nymphs) were collected and identified. Mean tick burdens were relatively heavy and the range in number of ticks per camel was very broad (6–173). Hyalomma dromedarii was the predominant tick species and accounted for 95.6% of the adult ticks. Other ticks found were H. marginatum subspp. and H. anatolicum excavatum. All nymphs collected were Hyalomma spp.In addition, the effect of ivermectin (Ivomec MSD AGVET) on tick burdens, when given subcutaneously at 0.2 mg/kg¹, was evaluated in nine camels in the date-palm plantation of Kibbutz Yahel in the Arava valley, Israel. Initial tick burdens on these camels (half-body tick counts) ranged from 20–105 ticks per camel. Seven camels from the same herd with half-body tick counts ranging from 40–107 ticks per camel were not treated and served as a control group. Ivermectin was not effective against Hyalomma tick infestation in camels under these conditions.     

Specificity and effects of host resistance on the African tickRhipicephalus zambeziensis (Acarina: Ixodidae)

Repeated infestations of the rabbit with adultRhipicephalus zambeziensis ticks resulted in a tenfold reduction in engorgement weight of adult ticks and a significant decline in engorgement weight of nymphs. There was no evidence of cross-resistance against the South African bont tick,Amblyomma hebraeum. Immunization of rabbits with crude extracts of whole-tick protein significantly influenced engorgement weights and oviposition rates of adultR. zambeziensis.Histological studies of tissues fromR. zambeziensis adults fed on resistant rabbits showed severe gut damage; this was associated with the binding of host IgG to mid-gut cells.