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Mixed infestation of nymphs and adults of Rhodnius prolixus Stal, 1859 and Panstrongylus geniculatus Latreille, 1811 was detected in 3 (15%) of 20 dwellings in El Guamito, an endemic focus of Chagas disease in Lara State, Venezuela. In one of the houses, both species were positive for Trypanosoma cruzi: 14.3% (R. prolixus) and 20% (P. geniculatus ). The overall infection rate in 143 of 352 R. prolixus was 16.1%. Parasites isolated from R. prolixus were identified as T. cruzi I by random amplified polymorphic DNA analysis. Dot-enzyme-linked immunosorbent assays of 36 R. prolixus showed that 58.3% of the R. prolixus had fed on humans. The gut contents of one fifth-instar nymph of P. geniculatus that was positive for T. cruzi also reacted with anti-human serum. A questionnaire was used to gather data on the demographic and socioeconomic characteristics of the population. An indirect immunofluorescent test, an indirect hemaglutination test, and an ELISA were used to detect the presence of antibodies against T. cruzi in 84 of 86 inhabitants and in 15.5% of people more than 20 years old. The relative risk (RR) of infection was greater in men than in women (RR = 1.61, 95% confidence interval = 0.54-4.80). Of the people more than 15 years old, 36.6% had no formal education. All respondents recognized triatomine bugs, but they did not relate them to Chagas disease transmission. A total of 85.7% of the houses were "ranchos" suitable for the colonization of triatomine bugs. The possible domiciliation of P. geniculatus and the implications of competition with R. prolixus for resources are discussed. Since there is no clear separation of food sources, abiotic factors such as microclimatic variation within houses may be critical to predict the outcome of the process of competition and potential domestication of this generally sylvatic species.

In selected localities of Ceské Budĕjovice and Ceský, Krumlov districts, well known by stable high incidence of tick-borne encephalitis (TBE) human cases but with low incidence of Lyme borreliosis, monitoring of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks was performed. Research was also aimed at the spread of I. ricinus to mountain areas of this region (National Park Sumava), as well as at investigating this tick for B. burgdorferi s.l. genospecies and TBE virus infection. Altogether 498 nymphs, 88 females and 11 males of I. ricinus from lower locations and 58 nymphs from mountain locations (760-1080 m above sea level) were tested by polymerase chain reaction. In lower locations total prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks was 35%. Single infection of Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto (s.s.) was found in 59, 50 and 63 ticks, respectively (i.e. in 12.8, 11.2 and 14.1%). Double infection was found in 42 ticks (6.0%) and triple infection in three ticks (0.4%). The high frequency of B. burgdorferi s.s. exceeds the as yet reported occurrence in Central Europe. These circumstances are discussed. In mountain locations B. afzelii was found in five ticks, that including two co-infection with B. garinii, in elevations of 762 m and 1024 m above sea level, respectively. This fact signals a real danger of human infections in a region that was previously deemed to be without risk. Moreover, this region is more and more the target destination of tourist activities. The results also suggest that the penetration of infection can be rapid and formation and establishment of natural focus of Lyme borreliosis might be rather quick.

Experiments were undertaken to determine the mode of transmission to cattle of an unnamed Babesia sp. by Hyalomma anatolicum anatolicum, Hyalomma detritum, Hy. rufipes koch, Haemaphysalis longicornis and Boophilus microplus. The unnamed Babesia species designated Babesia U sp. was isolated by infesting cattle with nymphs from female Hy. a. anatolicum ticks collected from Xinjiang province. Adults of laboratory reared Hy. a. anatolicum, Hy. detritum and Hy. rufipes koch were infected with Babesia U sp. by feeding on infected cattle, isolated with nymphal ticks of Hy. a. anatolicum derived from females collected from field. The experiments revealed that Hy. a. anatolicum was capable of transmitting Babesia U sp. transovarially in larval (2 of 4 calves), nymphal (6 of 6 calves) and adult (3 of 8 calves) stages, with prepatent periods of 16, 12, and 8 days, respectively, and that this Babesia was also transovarially transmitted by both the nymphal and adult stages of Hy. detritum and Hy. rufipes. Attempts to transmit this Babesia U sp. transovarially with Hae. longicornis and B. microplus, and transstadially with Hyalomma spp., were carried out, and the results proved to be negative.

American canine hepatozoonosis is an emerging, tick-transmitted infection of domestic dogs caused by a recently recognized species of apicomplexan parasite, Hepatozoon americanum. The known definitive host of the protozoan is the Gulf Coast tick, Amblyomma maculatum. Presently recognized intermediate hosts include the domestic dog and the coyote, Canis latrans. Laboratory-reared larval or nymphal A. maculatum can be infected readily by feeding to repletion on a parasitemic intermediate host; sporogony requires 35-40 days. Transmission of infection to the dog has been produced experimentally by oral administration of mature oocysts or oocyst-containing ticks. Canine disease follows experimental exposure in 4-6 weeks and is characterized by systemic illness, extreme neutrophilic leukocytosis, muscle and bone pain, and proliferation of periosteal bone. Histopathological findings include multifocal skeletal and cardiac myositis associated with escape of mature merozoites from within the host-cell environment. There is also rapid onset of periosteal activation and osteogenesis and, less frequently, glomerulopathy and amyloidosis. Sequential stages of development of H. americanum in both the dog and the tick have been elucidated. Gamonts potentially infectious to ticks have been observed in peripheral blood leukocytes of the dog in as few as 28 days after exposure to oocysts. Young coyotes experimentally exposed to a canine strain of H. americanum acquired disease indistinguishable from that of similarly exposed young dogs.

Groups of nymphal Rhipicephalus appendiculatus Muguga, having a mean of 1 or 9 Theileria parva Muguga-infected salivary gland acini per tick, were kept under quasi-natural conditions at an altitude of 1950 m or 20 degrees C at a relative humidity of 85% in the laboratory and their survival and infection prevalence and abundance determined over time. Theileria parva infections for both categories of ticks survived in the nymphal ticks for 50 or 26 weeks post salivary gland infection under quasi-natural or laboratory conditions respectively. There was a distinct decline in infections in the more heavily infected nymphae under both conditions of exposure, reflecting an apparent density dependence in parasite survival. Nymphal ticks having an average infection level of 1 infected salivary gland acinus per tick, survived for up to 69 or 65 weeks post-repletion under quasi-natural or the laboratory conditions respectively. Nymphae having an average infection level of 9 infected salivary gland acini per tick survived for a similar duration under each of the 2 conditions. The infection level of 9 infected salivary gland acini per tick did not seem to significantly affect the survival of the tick vector compared to those having an average of 1 infected salivary gland acinus per tick.

Juvenile freshwater parasitic nematodes Rhabdochona denudata honshuensis Moravec and Nagasawa, 1989 and Rhabdochona coronacauda Belouss, 1965 (Spirurida: Thelazioidea: Rhabdochonidae) were found in mayfly nymphs collected in a mountain stream in Japan. Considering the relative density of mayfly nymphs, nematode prevalence, and intensity of parasitism, Ephemera strigata Eaton and Potamanthus formosus Ulmer (Ephemeroptera: Ephemeridae) are frequent natural intermediate hosts for R. d. honshuensis in this locality. The intermediate host of R. coronacauda also is the E. strigata nymph.

An obscure parasitic fungus, Basidiolum fimbriatum, was found on Amoebidium parasiticum (Amoebidiales) associated with Caenis sp. (mayfly) nymphs, during a survey of gut fungi (Trichomycetes) from a small stream in northeastern Kansas, USA. The hindguts of the nymphs harboured a species of Legeriomycetaceae and Paramoebidium sp. This is the first report of the ectocommensal protozoan, A. parasiticum, associated with the gills of Caenidae (Ephemeroptera), and of B. fimbriatum in the 142 years since its original documentation from Wiesbaden, Germany. B. fimbriatum is recorded from two midwestern USA states (Kansas and Iowa) and the morphological and developmental features of the parasite on its host are compared with Cienkowski's original observations and interpretation. B. fimbriatum is characterized as a parasitic fungus possessing merosporangia that from on a simple pyriform thallus that penetrates and consumes its host via a haustorial network. The hypothesis that B. fimbriatum is most closely related to members of the order Zoopagales sensu Benjamin (1979) is proposed. The importance of future collections and molecular-based phylogenetic approaches to place this parasitic fungus within a current system of classification are highlighted.

A hypothesis for the independence of acts of infection in ticks with pathogens from each other in mixed infection has been tested on the basis of the data available in the literature. In most cases the number of ticks with mixed infection is consistent with the independence hypothesis and fails to reinforce the statement that there is antagonism of pathogens. But in some reports the number of mixed-infected ticks is 3-13 times higher than the calculated number according to the accepted hypothesis. These facts suggest that the natural tolerance of ticks to pathogens due to primary infection may be reduced. Other possible reasons for differences in the actual and calculated number of mixed infected ticks are also discussed.

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., (Copyright 2002 Elsevier Science B.V.)

A Babesia species has been identified and shown to be transmitted by Hyalomma anatolicum anatolicum in China. When larvae, nymphs and adults developed from engorged females H. a. anatolicum collected from cattle in Xinjiang province were infested onto the Babesia-free calves, piroplasms of Babesia sp. were seen in blood smears from cattle infested with nymphs (2 calves), but not from the calves infested with larvae (1 calf or adult ticks (2 calves). This Babesia sp. proved to be of low virulence, causing 3% parasites which lasted 3-4 days.

Phenotypic expression of trans-sialidase (TS) by Trypanosoma cruzi (Silvio strain) is restricted to a subpopulation (20-30%) of broad trypomastigotes (TS+ parasites), while the larger subpopulation of slender trypomastigotes lacks TS expression (TS- parasites). Rhodnius prolixus nymphs were infected with fractionated T. cruzi parasite populations (Silvio strain). Bugs ingesting TS- parasites produced significantly higher parasite loads than bugs ingesting either TS+ parasites or unfractionated populations. The addition of 1.0 microg of TS monoclonal antibody (TCN-2)/ml of blood meal to TS+ populations increased parasite populations to levels comparable to those of TS- populations. In contrast, the addition of exogenous TS to TS- parasite blood meals significantly reduced parasite loads to levels comparable to bugs ingesting TS+ parasites. These results suggest that T. cruzi trypomastigote polymorphism may serve to enhance survival in different host environments.

Laboratory-reared larval Gulf Coast ticks (GCTs) (Amblyomma maculatum) were exposed experimentally and found to acquire Hepatozoon americanum infection while feeding on parasitemic dogs. These ticks supported gamogonic and sporogonic development of the apicomplexan, and oocysts from newly molted nymphs were infectious for a dog. Other nymphs from this cohort that were allowed to feed on a blood-parasite naive sheep molted normally; the resulting adult ticks contained oocysts that were infectious for another dog. Merogonic development of H. americanum in the dogs and the resulting lesions/disease appeared similar, irrespective of whether infectious oocysts were derived from nymphal or adult ticks that acquired infection as larvae. In the system previously known, nymphal ticks acquire infection and adults harbor infective oocysts, which vertebrate hosts ingest. Given that larval A. maculatum can acquire infection and nymphs can harbor viable oocysts as demonstrated by this study, the potential variety of vertebrate hosts that can alternate with GCTs in maintaining an endemic cycle is considerably expanded.

Ixodes spinipalpis maintains Borrelia bissettii spirochetes in Colorado in a cycle involving wood rats and deer mice. This tick has been described as nidicolous, remaining either attached to its rodent hosts or in the rodent nest. Nidicolous ticks pose little risk of pathogen transmission to humans if they do not actively quest for hosts. To investigate the questing potential of I. spinipalpis, sentinel mice were placed in an area where I. spinipalpis had been commonly found on wood rats and deer mice. Concurrently, wild rodent populations were trapped and analyzed for Lyme disease spirochetes, the agent of human granulocytic ehrlichiosis (aoHGE), and Babesia microti. A total of 122 I. spinipalpis larvae and 10 nymphs were found on 19% of 244 sentinel mice. In addition, 4 sentinel mice became infested with Malaraeus telchinus or Orchopeas neotomae fleas. Questing I. spinipalpis were positively associated with woody shrubs and negatively associated with sunny and grassy areas. Four sentinel mice became infected with aoHGE after having been fed upon only by I. spinipalpis larvae. One sentinel mouse became infected with B. bissettii after having an I. spinipalpis nymph feed on it, and one sentinel mouse became coinfected with aoHGE and B. bissettii after it was fed upon by a single I. spinipalpis nymph. These sentinel mouse conversions suggest the possibility that the aoHGE is transovarially transmitted by I. spinipalpis, and that I. spinipalpis is capable of simultaneously transmitting B. bissettii and the aoHGE. The findings that I. spinipalpis quest away from rodent nests and will attach to and infect sentinel mice may be of public health importance. It suggests the potential transmission of the agents of human granulocytic ehrlichiosis and Lyme disease to other hosts by I. spinipalpis, in regions of the western United States where Ixodes pacificus is not found.

Alterations in host phenotype induced by parasitic infection are often interpreted as either host or parasite adaptations, depending on which of the two appears to benefit. Mermithid nematodes typically castrate their insect hosts and, therefore, any change in host behavior has no further fitness consequences for the host; the adaptive value of the modified behavior must be assessed with respect to parasite fitness only. In a New Zealand stream, mermithid-infected nymphs of mayflies in the genus Deleatidium were disproportionately represented in drift samples compared with benthic samples, suggesting that infection by mermithids results in an increased tendency to drift. Drifting mayflies face a higher predation risk from trout, and the mermithid nematodes they harbor die if ingested by a fish. The change in mayfly behavior induced by mermithids thus appears to have negative fitness effects for the parasite, and one possible explanation for this phenomenon is that it is a nonadaptive, pathological side effect of infection.

The incidence of first contact with the protozoan Theileria parva was determined in three traditional cattle herds in the Southern Province of Zambia in 1995 and 1996. The majority of first contacts occurred during the dry season in June, July and August, at a time of nymphal activity and in the absence of Rhipicephalus appendiculatus adults, indicating that larva to nymph transmission plays a more prominent role than nymph to adult transmission under the prevailing conditions.

Mice were tolerant to tick bites during three infestations with nymphs of Ixodes ricinus infected with Borrelia burgdorferi sensu stricto. To determine whether tick bites influence the immune response against B. burgdorferi, we examined the production of cytokines IL-4 and IFN-gamma by lymph node cells of BALB/c mice and IL-4 deficient BALB/c mice after tick inoculation versus syringe inoculation of B. burgdorferi. We also measured IgG2a anti-borrelial antibodies and total IgE in these mice. Results showed that BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation of B. burgdorferi. IL-4 deficient mice produced a Th1 immune response in both cases. IL-4 produced following tick bites greatly decreased the production of anti-borrelial IgG2a antibodies by comparison with the production of anti-borrelial IgG2a antibodies produced following syringe injection of B. burgdorferi.

Experiments were conducted to determine the effects of whitefly parasitoids on the cuticular lipid composition of the silverleaf whitefly, Bemisia argentifolii Bellows and Perring [=sweetpotato whitefly, Bemisia tabaci (Gennadius), Biotype B] nymphs. The cuticular lipids of B. argentifolii nymphs that had been attacked by parasitic wasps, either Eretmocerus mundus Mercet or Encarsia pergandiella Howard, were characterized by capillary gas chromatography and CGC-mass spectrometry and the results compared with the cuticular lipids of unparasitized nymphs. Previous studies with B. argentifolii nymphs had shown that wax esters were the major components of the cuticular lipids with lesser amounts of hydrocarbons, long-chain aldehydes, and long-chain alcohols. No appreciable changes in lipid composition were observed for the cuticular lipids of E. pergandiella-parasitized nymphs as compared to unparasitized controls. However, the cuticular lipids from nymphs parasitized by E. mundus contained measurable quantities of two additional components in their hydrocarbon fraction. Analyses and comparisons with an authentic standard indicated that the two hydrocarbons were the even-numbered chain length methyl-branched alkanes, 2-methyltriacontane and 2-methyldotriacontane. The occurrences and possible functions of 2-methylalkanes as cuticular lipid components of insects are discussed and specifically, in regard to host recognition, acceptance, and discrimination by parasitoids. Published 2000 Wiley-Liss, Inc.

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.

Studies were made on the development of Babesia gibsoni in the midgut of the nymphal stage of the tick, Rhipicephalus sanguineus. Six hr after repletion, merozoites of B. gibsoni, free of erythrocytes, were observed in the midgut contents of the ticks. After that, within 24 hr, those merozoites were transformed into ring-forms which were relatively large ring 1-2 microns in diameter. Later, the ring forms developed into spherical forms which were somewhat elliptical in shape and 3-4 microns in diameter. Within 2-4 days, bizarre forms (5-6 microns in diameter) developed into elongated forms (5-6 microns in length). About 5-6 days after repletion, large round or elliptic zygotes (7-9 microns in diameter) were observed in the ticks gut.

Euryconema brevicauda n. sp. parasitizing the mole cricket Neocurtilla claraziana found in Buenos Aires province, Argentina, is described and illustrated. This species is characterized by the male having 3 pairs of genital papillae, 1 pair preanal and 2 pairs postanal, and a short, conical-shaped tail.

Experimental transmission of Hepatozoon americanum to dogs was attempted with four ixodid ticks, viz., Rhipicephalus sanguineus, Amblyomma americanum, Amblyomma maculatum and Dermacentor variabilis. Ticks that dogs ingested included some that were laboratory-reared and experimentally fed as nymphs on a dog with naturally occurring hepatozoonosis; other ticks were collected as replete or partially engorged larvae, nymphs and adults from dogs that had hepatozoonosis and natural infestations of ticks. Whole ticks used to expose susceptible dogs orally were partially dissected to help release oocysts. Among eight dogs exposed, only the three that were fed A. maculatum adults experimentally acquisition fed as nymphs became infected. Dogs developed elevated body temperature and other evidence of clinical disease starting 4 weeks after exposure. 'Cysts' typical of H. americanum were found in skeletal muscle when samples were first examined 5 weeks after dogs ingested ticks, and parasites were also observed in peripheral blood smears at approximately the same time. Our study demonstrates that A. maculatum nymphs can acquire H. americanum by feeding on a parasitemic dog and that transstadial transmission of the protozoan occurs, with dogs acquiring infection when they ingest newly molted adult ticks.

Thirteen species or strains of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis were tested in vitro against unfed and engorged larvae, nymphs, and adults of the black-legged tick, Ixodes scapularis. Nematodes were pathogenic to engorged female ticks, but not to unfed or engorged larvae, nymphs, males, and unfed females. Steinernema riobravis (355) and Heterorhabditis megidis (M145) killed ticks most rapidly, with mean day of death postinfection of 2.5 and 3.5 days, respectively. However, all nematode strains and species were lethal to engorged female ticks within 7.5 days. Development of juveniles and reproduction of nematodes did not occur inside the tick cadavers, because no infective juveniles (IJs) emerged from cadavers after 40 days, and no nematode life-cycle stages other than the primary IJs could be isolated from cadavers. Egg masses were produced by 4 of 702 nematode-infected, engorged females. Egg masses produced were not significantly smaller than masses produced by control ticks; however, only 1 of the 4 egg masses hatched, whereas all of the egg masses produced by the control ticks hatched. Entomopathogenic nematodes may be useful as an alternative management method for I. scapularis populations, and may be more acceptable than acaricidal chemicals for use in infested areas.

Four hundred four patients with chronic Chagas' disease were treated with itraconazole (6 mg/kg of body weight/day for 120 days), allopurinol (8.5 mg/kg of body weight/day for 60 days), or with a placebo of pure starch. Patients were monitored over a period of four years by clinical examination, serology, xenodiagnosis, hemoculture, and electrocardiogram. Drug tolerance was good, with only four treatments discontinued due to side effects that subsided after suspension of treatment. Parasitologic cure was evident in 44% of the those treated with allopurinol and 53% of those treated with itraconazole, and the electrocardiographic evaluation showed normalization in 36.5% and 48.2%, respectively, of patients with chronic or recent cardiopathy.

Ticks were collected at irregular intervals from December 1995 to November 1996 from wildlife on Mtendere Game Ranch in the Chisamba District of Central Province, Zambia. Total collections were made from two species of ground-nesting birds and 20 species of small and large mammals. Thirteen species/subspecies of ixodid ticks were recovered. Rhipicephalus appendiculatus was the most abundant, followed by Boophilus decoloratus and Rhipicephalus evertsi evertsi. Small numbers of immature ticks of only a few species were collected from the birds and rodents. The lagomorphs carried large numbers of predominantly immature R. appendiculatus. Most of the ungulates harboured several tick species and had high infestations of R. appendiculatus. The seasonal abundances of Amblyomma variegatum, B. decoloratus, R. appendiculatus and R. evertsi evertsi were determined.

Antigens derived from partially engorged nymphs of Hyalomma anatolicum anatolicum were used in immunizing crossbred (Bos indicus x Bos taurus) cattle against larval, nymphal and adult H. a. anatolicum and H. dromedarii. The cattle were either infected with Theileria annulata at low parasitaemia or were uninfected. Whole nymphal extract (WNE), nymphal membrane antigens (NMA) and nymphal soluble antigens (NSA) were used for immunization. The group immunized with WNE showed significant and better rejection of H. a. anatolicum ticks as compared to calves immunized with either NMA or NSA. The moulting rates of both engorged larvae and nymphs remained unaffected. Nymphs which engorged on the immunized calves were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of Theileria infections in the resulting adult ticks from immunized and unimmunized Theileria infected cattle. These ticks also transmitted fatal theileriosis to susceptible calves.

As a first measure of Chagas' disease control in Brazil with chemical elimination of the most important vector of the disease, Triatoma infestans was removed. Attention is now being paid to Triatoma sordida and Panstrongylus megistus. That species can eventually be found inside houses, as happened with the specimens we examined from Bernardino de Campos and Sete Barras, all of them infected by Trypanosoma cruzi. These data suggest that a better knowledge about the behavior that species is needed to introduce changes in the control measures.

Significant increases in serum globulins (alpha 1, alpha 2, beta and gamma) were observed in mountain leopard tortoises (Geochelone pardalis) after they had been immunized with nymphal homogenates of Amblyomma marmoreum. There was a concomitant significant increase in the numbers of leukocytes (lymphocytes, basophils, monocytes and eosinophils). Resistance to nymphal-challenge infestations was manifested by reduced feeding time, lower engorgement masses, and significantly fewer (P < 0.0001) numbers of nymphs that moulted. These findings are contrary to the generally reported phenomenon, that ticks do not induce resistance in their natural hosts.

From January 1986 to February 1994, 563 xenodiagnosis (XD) were applied in 563 chronic chagasic patients from different areas of Brazil; 292 were women and 271 were men between 6 and 89 years (average: 41.4 +/- 14.7 years). To each XD 40 nymphs on the 4th stage were used: 20 from Panstrongylus megistus (Pm) and 20 from Triatoma infestans (Ti) in fast, during at least 14 days. The exam in each nymph was made 45 days after being applied on the patient, by observation in optical microscopy of the drugs and/or the grinded from the digestive tube. The results are: a) 205 (36.4%) positive XD, including 85 (15.1%) due only nymphs of Pm, 44 (7.8%) Ti and 76 (13.5%) Pm and Ti; b) positively in 4.9% of the nymphs from Pm and in 3.0% of the Ti nymphs examined. These results' analysis showed that the Pm nymphs were more sensitive that Ti's ones to the infection by Trypanosoma cruzi, increasing considerably the xenopositivity, independently from birthplace, sex or age of the patients. These results point out that to increase the efficacy of XD in chronic Chagas' disease, the exam must have more than a species of triatomine with different sensibilities to the T. cruzi infection, and in case of using one species on XD, Pm must substitute Ti.

Red veld rats (Aethomys chrysophilus) and bushveld gerbils (Tatera leucogaster) were trapped at monthly intervals, when possible, over a 2-year period, in the southern Kruger National Park, Mpumalanga Province. Forty-six specimens of each species were caught, euthenased and microscopically examined for fleas, lice, ticks and mites. Clear differences existed between the two rodent hosts in infestation intensity and also parasite species. The flea, Xenopsylla brasiliensis, commonly and exclusively utilized red veld rats, whereas Xenopsylla frayi was common and specific to bushveld gerbils. T. leucogaster were commonly infested with the lice Hoplopleura biseriata and Polyplax biseriata, while only a single A. chrysophilus hosted the louse, Hoplopleura patersoni. Red veld rats harboured small numbers of the immature stages of Haemaphysalis leachi/spinulosa and relatively large numbers of Rhipicephalus simus. The larvae of R. simus were irregularly collected from February to September and the nymphs from March to November. Bushveld gerbils hosted fewer ticks than did the rats, with a single specimen of H. leachi/spinulosa and low numbers of immature Hyalomma truncatum, the latter erratically present from June to October. Miles were abundant on both rodent hosts, A. chrysophilus hosting 13 species in six families, and T. leucogaster hosting 12 species representing seven families, with clear differences in mite assemblages between the two rodents. As the rats and gerbils were collected from the same trap lines at the same times, the differences in species composition and infestation intensity of their parasites, suggest that immunological behavioural or other segregating mechanisms are in operation to maintain discrete parasite assemblages.

A comparison was made of the agglutination and lysis of three strains of Trypanosoma cruzi in gut extracts and hemolymph of Rhodnius prolixus and the results obtained were correlated with the success or failure of the parasite strain to infect the digestive tube or to survive in the hemocel after inoculation. Both T. cruzi strains Dm28c and Cl urine 35 days after feeding with parasites. Concomitantly, both of these strains were agglutinated but not lysed by the crop extracts. In contrast, T. cruzi Y strain parasites rapidly disappeared from the gut and showed no agglutination, but some lysis, in the crop extract. Following inoculation into the hemocel, only the Cl strain survived at high levels and was also the only strain agglutinated significantly in the hemolymph. Both Dm28c and Y strains rapidly disappeared from the hemocel with the former parasite being removed more slowly than the latter, probably due to clearance by the cellular defenses. The rapid clearance of the Y strain was correlated with the presence of a high titer lysin in the hemolymph. Subsequent experiments using FITC-labeled lectins and FACS to probe the carbohydrates on the parasite surfaces showed significant differences between the three strains. Thus, only Dm28c was stained strongly by Arachis hypogea (PNA) lectin, indicating the presence of galactose/N-acetylgalactosamine residues, and Dm28c and Y strains by Phytolacca lectin for N-acetyl glucosamine moieties. Finally, the fact that, in contrast to Dm28c and Y, the Cl strain strongly interacted with Triticum vulgaris (WGA) but not with Phytolacca lectin may be due to the presence of N-acetyl neuraminic acid residues on these organisms. These surface carbohydrate differences may be correlated both to the behavior and agglutination variations between the three strains recorded in this work.

The transmission of Borrelia burgdorferi to deer mice (Peromyscus maniculatus) by Ixodes pacificus nymphs was investigated experimentally. Deer mice were exposed to infected nymphs for 24, 48, or 72 hr, or until ticks had fed to repletion (> or = 96 hr). Infection status of hosts was assessed 4 wk later by culture of ear-punch biopsies in BSK II medium and by indirect immunofluorescence. Eight mice exposed to ticks for 24 hr did not become Infected. In contrast, infection was acquired by 1 of 9 (11%), 2 of 8 (25%), and 8 of 10 (80%) mice exposed for 48, 72, and > or = 96 hr, respectively. Eight weeks after exposure to infected nymphs, the infectivity of 5 deer mice for I. pacificus larvae was assessed. Overall, 33% of I. pacificus larvae fed on these mice acquired and transstadially passed spirochetes. We conclude that most I. pacificus nymphs require 4 days or longer to transmit spirochetes to deer mice, and that larvae efficiently acquire and maintain spirochetes from mice that have been infected by tick-bite.

The estimation of two parameters in the transmission of Babesia microti by the tick Ixodes trianguliceps amongst small mammals, (1) the duration of infectivity in natural hosts and (2) the probability of transmission from an infected to a susceptible vole, is described. When B. microti was maintained by direct tick transmission, the probability of a complete cycle of transmission via the larval-nymphal and nymphal-adult transstadial routes was 1.0 and 0.71 respectively, but only if the larvae or nymphs had engorged, as distinct from feeding slowly, while the source parasitaemia exceeded 2 or 0.2% respectively, but had not yet passed the peak level. The duration of this condition for infectivity in voles infected by nymphal bites was only 1-4 days, whilst infections delivered by adult ticks barely reached the threshold level necessary for successful transmission. When syringe passage was introduced into the parasite maintenance schedule (a) the probability of transmission declined markedly and (b) the time-course of the parasitaemia was altered. If these parameter values are put into a simple model, together with field data on tick and host survival rates, it becomes apparent that additional factors, such as the highly aggregated distribution of ticks on their hosts, must account for the maintenance of B. microti at the levels seen in wild small mammal populations.

To determine whether protective immunity against the agent of Lyme disease may be expressed mainly within its tick vector prior to transmission, we passively immunized mice at various intervals after infected ticks had attached, and assayed such mice for evidence of spirochetal infection by xenodiagnosis one month after challenge. Groups of CD-1 mice were intraperitoneally infused with 0.5 ml of hyperimmune rabbit or mouse serum, reagents and quantities previously determined to protect against syringe-challenge with 10(6) low-passage JD1 spirochetes 12 hr after passive transfer. Comparison groups received normal rabbit serum or saline. All mice were protected from infection when infused no more than one day after infective ticks were allowed to attach. However, if infused three or five days post-tick attachment, 60-100% of the mice became infected. All mice became persistently infected when infused with saline or normal rabbit serum. We conclude that antibody is protective against tick-transmitted spirochetal infection only when passively administered before the spirochetes are deposited in the skin of the host. Ingested antibody may destroy spirochetes or interfere with activation and replication within the tick gut, or with dissemination to the salivary glands. Lyme disease vaccines may thus be uniquely effective because of the vulnerability of the spirochetal agent within its vector.

Intermediate and definitive host specificity of Rhabdochona canadensis in Nebraska were investigated. Mayfly nymphs Trichorythodes sp. and Caenis sp. were found to serve as experimental intermediate hosts. Development inside the nymphs required approximately 10 days, with the worms passing through 2 molts and then becoming encapsulated in the hemocoel as infective third-stage juveniles. Survey data revealed that only the red shiner Cyprinella lutrensis serves as definitive host for R. canadensis in nature. Laboratory infections of Notropis dorsalis, N. stramineus, and Fundulus zebrinus, all of which were uninfected in nature, were attempted to determine if observed specificity was due to physiological or ecological factors. Two individuals of N. dorsalis became infected with R. canadensis, but no development was observed. Both N. stramineus and F. zebrinus were incapable of becoming infected. Thus, definitive host specificity in this system seems to be mediated by both physiological and host ecological factors.

The overall prevalence of Theileria species, mainly, if not exclusively, T.annulata, in 901 Hyalomma detritum detritum collected from cattle in the Doukkala region of Morocco over a period of 2 years was 21.5%. The quantity of infection (number of sporoblasts per infected tick) followed the negative binomial distribution with between one and 250 sporoblasts per infected tick. Infected ticks were found in eight of fourteen areas examined whilst T.annulata was present in all fourteen. There were significant differences in both the prevalence and the quantity of infection between ticks collected from different farms, and between nymphs collected in the autumn from these farms, and moulted in the laboratory, and adults collected in the following summer. The prevalence, but not the quantity, of infection was higher in female than in male ticks. No correlations were established between infection of engorged nymphs and the breed, sex and Theileria piroplasm parasitaemia of the host animal. However, calves infected a greater proportion of nymphs than adult cattle and the heavier the infestation of nymphs on an animal, up to a plateau, the higher the prevalence of infection in those nymphs. There were no differences in infection between ticks moulted at 24 degrees C and 37 degrees C, after the engorged nymphs had been stored at 18 degrees C to simulate over-wintering.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ioxodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 degrees C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.

To better define the possible role of argasid ticks in the epidemiology of West Nile virus, adult Argas arboreus, A. persicus, and A. hermanni were fed through a membrane on fetal bovine serum containing 10(5.5) 50% tissue culture infective doses (TCID50)/ml of West Nile virus. The virus was detected for three and four days after feeding in A. persicus and A. hermanni, respectively. The virus titers then decreased to undetectable levels in both species. When the infective dose was increased to 10(6.2), virus was detected until days 6 and 8, respectively. In A. arboreus, virus titers in whole tick homogenates reached a peak of 10(4.0) on day 4 postfeeding and remained constant at 10(3.0) after day 6 throughout the 20- or 50-day observation periods. Virus was detected by isolation, indirect fluorescent antibody, and histochemical techniques in the salivary glands, ovaries, synganglia, and coxal fluids. Infected ticks successfully transmitted virus to clean chickens on day 20 postfeeding. No evidence of transstadial transmission from nymph to adult was detected. Larvae from experimentally infected females successfully transmitted virus to clean chicks and virus was recovered from F1 larvae. Venereal transmission was not detected. Virus was present in coxal fluids secreted by infected females after infective meals. This study demonstrates West Nile virus infection in experimentally infected A. arboreus ticks and documents horizontal and vertical transmission in this species.

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30 degrees C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.

Crimean-Congo hemorrhagic fever (CCHF) virus was transmitted from infected adult Hyalomma ticks to uninfected larval and nymphal Hyalomma ticks while cofeeding on a guinea pig host that did not have a detectable viremia. When tested after feeding with infected adults, three (0.8%) of 370 H. truncatum larvae contained detectable CCHF virus (mean virus titer 10(1.6) plaque-forming units [PFU]/tick). The virus was transmitted transstadially from infected larvae and was detected in 15 (1.2%) of 1,253 nymphs and 12 (0.1%) of 2,049 adults. Virus was recovered from 18 (1.9%) of 931 H. impeltatum nymphs, which originated from larvae that cofed with infected adults. After H. impeltatum nymphs cofed with infected adults, CCHF virus was detected in 21 (4.3%) of 449 (mean virus titer 10(1.7) PFU/tick) fed nymphs, but none of 886 adults tested after molt. Results of this study indicate that a small proportion of either larvae or nymphs may acquire CCHF infection while cofeeding on a host without a detectable viremia.

The wasp Hunterellus hookeri Howard parasitizes several species of ixodid ticks including Ixodes dammini Spielman, Clifford, Piesman & Corwin, the vector of Lyme disease spirochetes, Borrelia burgdorferi, in the northeastern United States. We detected the occurrence and evaluated the distribution of this wasp parasitoid in populations of I. dammini in Rhode Island. There has been no intentional introduction of a tick parasitoid recorded anywhere in Rhode Island; yet, we found this wasp at one of the six study sites (Prudence Island), where it parasitized 21 (n = 243) and 17% (n = 284) of nymphal I. dammini collected during 1988 and 1989, respectively. The proportion of nymphs parasitized was greatest during May (46%) and was less in June (18%), July (18%), and August (11%). In Rhode Island, the wasp was only found parasitizing ticks at the site with the highest (by a factor of 2) tick population, confirming similar observations in Massachusetts and New York. It is suggested that establishment as well as the distribution of H. hookeri depends upon a super abundant deer tick population. The usefulness of this parasitoid as a biological control agent is yet unknown.

Rhipicephalus simus was, for the first time, experimentally proven to be a transovarial vector of Babesia trautmanni of domestic pigs. The nymphal and adult progeny of experimentally infected female ticks transmitted the infection to 2 susceptible splenectomized pigs. Features of the infection included a prepatent period of 6-8 days post-tick infestation, a febrile reaction for 3 days and a maximum parasitaemia score of 15 (more than 6 parasites per 300 red blood cells). Other clinical signs in both pigs were mild inappetence and listlessness. Both pigs recovered without any antibabesial therapy.

Prevalence of triatomines and their infection by Trypanosoma cruzi, were studied during 1974-1981, in the Administrative Region of Campinas, São Paulo, Brazil. This investigation was continued (1982-1986), blood meal tests with bird, marsupial, rodent and human antisera having been added. Panstrongylus megistus, predominant in the region, whose winged instars are often found infected by T. cruzi in inhabited houses, displayed and alimentary eclecticism. From a sample of 7,785, 14.78 percent reacted positively with human antisera. A similar, but less marked behaviour was observed in Rhodnius neglectus. Triatoma sordida, the second most important species in the Region, was neither demonstrated to be feeding on humans nor to be infected by T. cruzi. Changes in their natural habitat probably caused sharp rises in T. arthurneivai populations in the human dwellings. The capture of three specimens of Microtriatoma borbai marks the first occurrence of this species in the State São Paulo. "Focus investigation" in areas in the surveillance phase can detect triatomines in the process of domiciliation.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.

Studies on vectors of Trypanosoma cruzi Chagas were conducted during 1982-85 as part of an investigation on the epidemiological status of American Trypanosomiasis in Trinidad, West Indies. A total of 148 reduviid bugs were collected from caves, armadillo nests and at light traps, comprising four sylvatic species and totalling 120 Panstrongylus geniculatus (Latreille), two P. rufotuberculatus (Champion), twelve Rhodnius pictipes Stal and fourteen Eratyrus mucronatus Stal. A significantly higher level of infection with T. cruzi was recorded for P. geniculatus (42.5%) compared to R. pictipes (8.3%). The other two bug species were not found to be infected. P. geniculatus seems to be the most important vector in the sylvatic cycle of transmission of T. cruzi in Trinidad.

Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina-specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization.

The effect of temperature on the transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum was studied. Variation in temperature (4-40 degrees C) had a significant effect on moulting rate of the ticks and transmission of theilerial parasites from nymphs to resultant adults. The temperatures above 40 degrees C and below 12 degrees C prevented moulting. Maximum infection levels were obtained in salivary glands of adult ticks when the infected engorged nymphs were incubated at 24-28 degrees C. The infection rate in salivary glands was assessed using a methyl green pyronin technique.