Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥10⁴ organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.     

Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus

Equi merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equi proteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileria spp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equi transitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equi stages in the midgut lumen and midgut epithelial cells of Boophilus microplus nymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equi in salivary glands of adult Boophilus microplus. In addition, quantification of B. equi in the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equi organisms in the midgut. In contrast, there was no conclusive evidence that B. equi EMA-1 was expressed in either the Boophilus microplus midgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equi EMA-2 in Boophilus microplus provides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesia proteins for testing transmission-blocking immunity.     

Specific immune responses are required to control parasitemia in Babesia equi infection.

Horses possessing a normal immune system and spleen often control infection caused by Babesia equi. However, splenectomized horses are unable to control B. equi infection and usually succumb to the infection. To investigate the role of the spleen in the control of B. equi infection in the absence of specific immune responses, two 1-month-old foals with severe combined immunodeficiency (SCID) and two age-matched normal foals were inoculated with B. equi. The SCID foals became febrile seven days postinoculation and developed terminal parasitemias of 41 and 29%. The SCID foals had greater than 50% decreases in indices of total erythrocytes, packed-cell volumes, and hemoglobin concentrations. Both SCID foals were euthanized in extremis at 10 days postinoculation. As expected, the serum of the SCID foals lacked detectable antibodies to B. equi antigens. In contrast, the normal foals inoculated with B. equi produced detectable anti-erythrocyte-stage parasite antibodies by 7 days and controlled clinical disease by 12 days postinoculation. Although SCID foals lack functional T and B lymphocytes, they do possess complement, macrophages, granulocytes, and natural killer cells, as well as a spleen. Therefore, the data indicate that specific immune responses are required to control B. equi parasitemia but are not required for erythrocyte lysis in infected horses. Furthermore, the spleen is not able to control B. equi parasitemia in the absence of specific immune responses to parasite antigens.     

Transovarial transmission efficiency of Babesia bovis tick stages acquired by Rhipicephalus (Boophilus) microplus during acute infection

The protozoan parasite Babesia bovis, a reemerging threat to U.S. cattle, is acquired by adult female ticks of the subgenus Boophilus and is transovarially transmitted as the kinete stage to developing larval offspring. Sporozoites develop within larvae and are transmitted during larval feeding on a bovine host. This study evaluated the efficiency of B. bovis infection within Rhipicephalus (Boophilus) microplus following acquisition feeding on acutely parasitemic cattle. Parasite levels were quantified in blood from experimentally infected cattle and within hemolymph and larvae derived from acquisition-fed female B. microplus. There was a positive correlation between blood parasite levels in acutely parasitemic cattle and kinete levels in the hemolymph of adult female Boophilus ticks following acquisition feeding; however, there was no relationship between kinete levels in females and infection rates of larval progeny. Boophilus microplus females that acquisition fed produced larval progeny with infection rates of 12% to 48%. Importantly, larvae derived from replete females with very low levels of kinete infection, as demonstrated by microscopy and PCR, had infection rates of 22% to 30% and transmitted B. bovis during transmission feeding. These data demonstrate that although hemolymph infection may be undetectable, transmission to larval progeny occurs at a level which ensures transmission to the bovine host.     

Dermacentor variabilis and boophilus microplus (Acari: Ixodidae): experimental vectors of Babesia equi to equids

The experimental vector competence of five laboratory-reared ixodid tick species representing three genera [Amblyomma americanum (L.), Boophilus microplus (Canestrini), D. andersoni Stiles, D. occidentalis Marx, and D. variabilis (Say)] for Babesia equi (Laveran 1901) was evaluated by delayed transfer of male ticks from infected to susceptible equids or by infesting the latter animals with adult ticks previously fed as nymphs on infected equids. After feeding for 5, 6, or 13 d on acquisition hosts, ticks were forcibly removed and held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 6, 12, or 27 d. Intrastadial transmission to susceptible ponies by D. variabilis males, and transstadial transmission to susceptible burros by B. microplus adults, was demonstrated by blood smear and indirect immunofluorescence serology. The data indicated that male D. variabilis and adult B. microplus, tick species that occur on equids in North America and, in the case of the latter tick, also extensively in tropical and subtropical regions of the world, may be competent natural vectors of B. equi     

Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae)

Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 10(9) infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 10(2.5) and 10(7) IE/ ml of blood. We compared stable fly Stomoxys calcitrans (L.) -borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.     

Persistently infected calves as reservoirs for acquisition and transovarial transmission of Babesia bovis by Rhipicephalus (Boophilus) microplus

Babesia bovis is a deadly disease of cattle resulting in severe economic losses in the vast regions of the world where it is endemic. If reintroduced into the United States, babesiosis would cause significant mortality in the naïve cattle population. In order to address the risk to U.S. cattle, it is essential to quantify the transovarial transmission efficiency in adult female Boophilus microplus ticks following acquisition feeding on persistently infected cattle. This study tested the hypothesis that infection rates are the same for larval progeny derived from females fed to repletion during persistent or acute infection. Increasing parasite levels during acute infection correlated with an increasing number of females harboring kinetes detectable in hemolymph (r = 0.9). The percent infected larvae ranged from 0 to 20% when derived from females fed to repletion on persistently infected calves and from 4 to 6% when derived from females fed to repletion during acute parasitemia. There was no significant difference in infection rates of larval progeny, implying that the risk associated with the introduction of either persistently infected or acutely infected cattle is equal. Parasite levels ranged from 2.4 × 10² to 1.9 × 10⁵ in 3-day-fed larvae derived from females fed to repletion on persistently infected cattle. One group of larvae failed to transmit the parasite, suggesting that a threshold level of parasites must be obtained by larval progeny via transovarial transmission in order for larvae to deliver sufficient parasites to infect a naïve host.Bovine babesiosis, also known as Texas cattle fever, is endemic to tropical and subtropical regions of the world including Central and South America, Asia, Australia, and Africa and is ranked as the most economically important arthropod-transmitted pathogen of cattle (1). The disease is caused by the tick-borne apicomplexan protozoon Babesia bovis and is characterized by anemia, fever, and, in severe cases, multiorgan failure resulting in death. Young calves are relatively resistant to severe disease and have the potential to recover but remain persistent carriers with no clinical signs of disease (8, 18). While parasite levels in the persistent state are often undetectable (4-6), ticks may still be capable of acquiring infection from these animals. Thus, persistently infected cattle are potentially an important facet in the maintenance of B. bovis in nature as well as in the introduction and spread of the parasite to nonendemic areas where competent vectors are present.Following outbreaks that resulted in devastating economic losses to the U.S. cattle industry, the major tick vectors of B. bovis, Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, were eradicated from the continental United States by 1943 (2, 7, 10). Today, there remains a quarantine zone along the border between Texas and Mexico that extends from Brownsville to Del Rio. In Mexico, both the parasite and vector remain prevalent, and acaracide-resistant Boophilus ticks are increasingly common (15-17). There is no serological testing of cattle within the quarantine zone, and therefore, movement of cattle is not restricted based on B. bovis infection status. Due to the increase in acaracide-resistant ticks and the lack of clinical signs in persistent cattle, the introduction of infected ticks and/or cattle into the United States is likely. The introduction of babesiosis into the previously unexposed cattle population outside the limits of the quarantine zone would result in significant mortality.When an adult female Boophilus tick feeds on an infected bovine host, the merozoite stage of B. bovis is acquired. Following gametogenesis and zygote formation within the lumen of the midgut, the kinete stage is released into the hemolymph of the female. The kinete stage can be detected in the hemolymph of the tick during migration from the midgut to ovaries. After invasion of the ovaries, kinetes are transovarially transmitted to developing larvae. Within developing larvae, B. bovis invades salivary glands and develops into infective sporozoites, which are subsequently transmitted when larvae commence feeding on a bovine host.Determining the efficiency of transmission is crucial to developing strategies to prevent the reintroduction of B. bovis into the United States. If the efficiency of transovarial transmission is equivalent in females acquiring the parasite from either acutely or persistently infected cattle, and should emerging acaracide resistance lead to the reestablishment of B. microplus in the United States, then the absence of serological screening of cattle entering the United States is a definite oversight. In the current study, we began to address this issue by examining the transovarial transmission efficiency of female B. microplus ticks fed to repletion on persistently infected calves. We hypothesized that infection rates of larval progeny from these females would be the same as infection rates of larval progeny from females fed to repletion during acute parasitemia. We examined hemolymph kinete levels in females by light microscopy and nested PCR, determined infection rates of transmission-fed larval progeny by nested PCR, and quantified parasite levels in transmission-fed larvae using real-time PCR.     

Ultrastructure of sporogony in Babesia equi in salivary glands of adult female Boophilus microplus ticks

The development of Babesia equi was studied in the salivary glands of adult female ticks, Boophilus microplus, using a transmission electron microscope (TEM). Engorged nymphs were obtained from splenectomized foals experimentally infected with B. equi and fed in the adult phase for 5 days on rabbits. Sporogony in B. equi involves the development of sporoblasts and sporozoites, which form from finger-like projections on the surface and through radial budding. Mature sporozoites (2.0 × 1.1 μm), typically pyriform, showed a polar ring, rhoptries, micronemes, nuclei, and mitochondria, and a high concentration of free ribosomes were observed from the 2nd day of the ticks, feeding on the rabbits. In general, sporogony of B. equi in the salivary glands of B. microplus showed similarities to the development of this parasite in species of Hyalomma, although with some significant differences in the sporozoite's dimensions. The results of this study indicate that B. equi is capable of multiplying in the salivary glands of adult female B. microplus, forming sporozoites with specialized organelles characteristic of the invasive form, and suggest that B. microplus can act as a natural vector of B. equi in endemic areas where there is no other probable source of infection or where it is the only tick species present on horses. Received: 5 February 1997 / Accepted: 14 May 1997     

Computer simulation of Babesia bovis (Babes) and B. bigemina (Smith & Kilborne) transmission by Boophilus cattle ticks (Acari: Ixodidae).

A computer model was developed to simulate the processes involved in transmission of the cattle fever parasites Babesia bovis (Babes) and Babesia bigemina (Smith & Kilborne) between cattle and Boophilus ticks. The model of Babesia transmission was combined with a dynamic life history model for population dynamics of the tick vectors, Boophilus microplus (Canestrini) and B. annulatus (Say). Epidemiological parameters and relationships in the model include the reduction in fecundity of infected ticks, rate of transovarial transmission, effect of cattle type and inoculation rate on infectivity of cattle, variation of infected cattle recovery rate with age of infection, inoculation rate, and species of parasite. Some parameters in the model were fitted by iterative simulations to produce realistic rates of Babesia infection in larval ticks. Comparisons of simulated and reported epidemiological data from one location in Australia indicated a reasonable level of validity for the model. Theoretical tick density thresholds for maintenance of Babesia in cattle and for inoculation of greater than or equal to 99.5% calves were determined by iterative simulations at 10 locations with B. microplus and six locations with B. annulatus. The model and transmission thresholds can serve as the basis for further simulation studies on strategies for control or eradication of babesiosis.     

Development of specific immunoglobulin Ga (IgGa) and IgGb antibodies correlates with control of parasitemia in Babesia equi Infection

In this study, the kinetics of specific immunoglobulin G (IgG) isotypes were characterized in Babesia equi (Theileria equi)-infected horses. IgGa and IgGb developed during acute infection, whereas IgG(T) was detected only after resolution of acute parasitemia. The same IgG isotype profile induced during acute infection was obtained by equi merozoite antigen 1/saponin immunization.     

A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites.

Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses.     

Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi.

Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.     

Infection of immunodeficient horses with Sarcocystis neurona does not result in neurologic disease

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.     

During Trypanosoma cruzi Infection CD1d-Restricted NK T Cells Limit Parasitemia and Augment the Antibody Response to a Glycophosphoinositol-Modified Surface Protein

Trypanosoma cruzi is a protozoan parasite that chronically infects many mammalian species and in humans causes Chagas' disease, a chronic inflammatory disease. The parasite expresses glycophosphoinositol (GPI), which potently stimulates interleukin 12 (IL-12) production. During T. cruzi infection IL-12, and possibly GPI, might stimulate NK T cells to affect the protective and chronic inflammatory responses. Here we report that during T. cruzi infection CD1d-restricted NK T cells are stimulated as NK T-cell-deficient mice have greater parasitemia. Furthermore, during T. cruzi infection the percentages of NK T cells in the liver and spleen become decreased for prolonged periods of time, and in vitro stimulation of NK T cells derived from livers of chronically infected mice, compared to uninfected mice, results in increased gamma interferon and IL-4 secretion. Moreover, in NK T-cell-deficient mice the chronic-phase antibody response to a GPI-modified surface protein is decreased. These results indicate that, during the acute infection, NK T cells limit parasitemia and that, during the chronic phase, NK T cells augment the antibody response. Thus, during T. cruzi infection the quality of an individual's NK T-cell response can affect the level of parasitemia and parasite tissue burden, the intensity of the chronic inflammatory responses, and possibly the outcome of Chagas' disease.     

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.     

Conservation of transmission phenotype of Anaplasma marginale (Rickettsiales: Anaplasmataceae) strains among Dermacentor and Rhipicephalus ticks (Acari: Ixodidae)

Before the eradication of Boophilus ticks from the United States, Rhipicephalus (Boophilus) microplus (Canestrini) and Rhipicephalus (Boophilus) annulatus (Say) were important biological vectors of the cattle pathogen Anaplasma marginale Theiler. In the absence of Boophilus ticks, A. marginale continues to be transmitted by Dermacentor ticks. However, a few U.S. strains are not transmissible by Dermacentor andersoni Stiles, Dermacentor variabilis (Say), or both, raising the question of how these strains evolved and how they are maintained. We hypothesize that the U.S. non-Dermacentor-transmissible strains of A. marginale were formerly Boophilus-transmitted strains that have been maintained by a combination of persistent infection and mechanical transmission since the eradication of their biological vector from the United States. To test this hypothesis, we attempted to transmit a well-documented non-Dermacentor-transmissible A. marginale strain (Florida), by using D. andersoni and the two Boophilus species that formerly occurred in the United States. For comparison, we examined tick-borne transmission of a strain of A. marginale (Puerto Rico), which has previously been shown to be transmissible by both D. andersoni and B. microplus. All three species of tick transmitted the Puerto Rico strain, and immunohistochemical (IHC) analysis confirmed the presence ofA. marginale colonies in their salivary glands. All three tick species failed to transmit the Florida strain. Although both D. andersoni and B. microplus acquired transient midgut and salivary gland infections after acquisition feeding, we were unable to detect colonies of the Florida strain in the salivary glands with IHC. This demonstrates that the transmission phenotype ofA. marginale strains is conserved among tick species, and it suggests that the failure of the Florida strain to be transmitted by ticks is related to a general inability to efficiently invade or replicate in tick cells, rather than to a failure to invade or replicate in cells of a specific tick species.     

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on na?ve mice, all of which became infected as shown by culture of ear biopsy specimens. Na?ve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.     

Infection of Immunodeficient Horses with Sarcocystis neurona Does Not Result in Neurologic Disease

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 × 10⁵ sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 × 10⁸ merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.     

Experimental Chagas'' disease: kinetics of lymphocyte responses and immunological control of the transition from acute to chronic Trypanosoma cruzi infection.

The responses of spleen cells from mice infected with Trypanosoma cruzi to stimulation with T (concanavalin A and phytohemagglutinin) or B (lipopolysaccharide) cell-specific mitogens were monitored during the acute, transition, and chronic states of the disease. A marked reduction in the responses of infected mouse cells with respect to those of uninfected animals was observed during the acute stage, regardless of whether or not the infective dose was lethal. Reduced or absent responses were recorded with suboptimal, optimal, and supraoptimal concentrations of the mitogens. Normal levels of responsiveness to concanavalin A, phytohemagglutinin, and lipopolysaccharide were observed during the chronic stage of the disease. The trend of return to normal responses was initiated around day 40 after infection with 25 parasites. At this time, a marked decline in parasitemia levels, cessation of mortality, and disappearance of visible signs of disease began to be observed defining the transition stage that precedes establishment of chronicity. T cell levels of the spleen were markedly reduced during the acute period and returned during the chronic phase. Instead, absolute levels of B cells were significantly increased during the acute period but also normalize in the chronic phase. Immunosuppression of chronically infected mice with cyclophosphamide led to a temporary return to acute infection-type conditions, even in animals with undetectable levels of parasitemia before treatment. These results suggest that reduced T cell responses during acute experimental Chagas' disease might in part to be due to depletion of the T cell compartment. Decreased B cell responses in the presence of significant numbers of B lymphocytes implies a suppressive phenomenon, B cell alteration, or a combination of both possibilities. Recrudescence of the disease after immunosuppression with cyclophosphamide suggests that immunological mechanisms play an important role, not only in the gain of control over T. cruzi infected by the host but also in the maintenance of the chronic status.     

Accelerated transmission of Lyme disease spirochetes by partially fed vector ticks.

To determine how rapidly Lyme disease spirochetes (Borrelia burgdorferi) can be transmitted by partially fed vector ticks (Ixodes dammini), attached nymphs were removed from their hosts at various intervals post-attachment and subsequently permitted to re-feed to repletion on noninfected mice. We confirm previous reports that ticks deposit Lyme disease spirochetes in the skin of their hosts mainly after 2 days of attachment. Those that have been removed from a host within this interval can reattach and commence feeding. Spirochete-infected nymphs that have previously been attached to a host for 1 day become infectious to other hosts within another day. Noninfected nymphs acquire infection from spirochete-infected hosts within a day of attachment and become infectious to other hosts 3 to 5 days later. Virtually all ticks transmitted infection when reattaching after first feeding for 2 days. We conclude that partially fed nymphal ticks transmit spirochetal infection more rapidly than do ticks that have never been attached to a host and that infected ticks become infectious before they molt.