Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study.

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cell-rich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

The bovine spleen: interactions among splenic cell populations in the innate immunologic control of hemoparasitic infections.

Over the past several years, innate immunity has been recognized as having an important role as a front-line defense mechanism and as an integral part of the adaptive immune response. Innate immunity in cattle exposed to hemoparasites is spleen-dependent and age-related. In this review, we discuss general aspects of innate immunity and the cells involved in this aspect of the response to infection. We also provide examples of specific splenic regulatory and effector mechanisms involved in the response to Babesia bovis, an important tick-borne hemoparasitic disease of cattle. Evidence for the regulatory and effector role of bovine splenic monocytes and DC both in directing a type-1 response through interaction with splenic NK cells and γδT-cells will be presented.

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells.

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites.

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.

Prospects for recombinant vaccines against Babesia bovis and related parasites.

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen.

The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-gamma. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-gamma and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.

Age-related innate immune response in calves to Babesia bovis involves IL-12 induction and IL-10 modulation.

There is a strong innate immunity in calves to infection with Babesia bovis. Interleukin (IL)-12 and IL-10 have been shown in vitro to be important immunoregulatory cytokines. Here we demonstrate in vivo that the protective innate response in young calves to infection with virulent B. bovis involves the early appearance of IL-12 and interferon-gamma (IFN-gamma) transcripts in the spleen. In contrast, IL-12 and IFN-gamma mRNA expression in the spleens of adult cattle that succumbed to the infection was delayed and depressed and occurred within the context of IL-10 expression. Also in contrast with calves, there was no detectable antibody response before death in adults. A vigorous CD8+ T-cell expansion occurred in the spleens of both calves and adults.

IL-4 and IL-10 inhibition of IFN-gamma- and TNF-alpha-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites.

The requirement for IFN-gamma and/or TNF-alpha as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-gamma and TNF-alpha unless the monocytes taken ex vivo were producing TNF-alpha endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-gamma-dependent. There was no evidence for endogenous synthesis of TNF-alpha in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-alpha mRNA in MDM. However, while merozoites plus IFN-gamma induced TNF-alpha mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.

The age-related immunity in cattle to Babesia bovis infection involves the rapid induction of interleukin-12, interferon-gamma and inducible nitric oxide synthase mRNA expression in the spleen.

Young calves possess a strong innate immunity against Babesia bovis infection that lasts for approximately 6 months after birth and is abrogated with the removal of the spleen. This immunity is characterized as cellular involving a soluble mediator. Nitric oxide has been implicated by virtue of its babesiacidal affects in vitro, but questioned to be as effective in vivo, due to its ability to downregulate type-1 immunity. Spleen cells were obtained from 4-month-old calves and adult steers and processed for monitoring cytokine and inducible nitric oxide synthase (iNOS) mRNA expression during the response to initial B. bovis infection. The data provided evidence of a transient role for nitric oxide in innate immunity, characterized by brief iNOS induction in the spleen of calves that was not detectable in the spleens of adults. The iNOS message followed the early induction of interleukin (IL)-12 and interferon (IFN)-gamma message in calves. The induction of IL-12 and IFN-gamma message in adults was delayed until IL-10 message was induced. Transformation growth factor-beta mRNA expression levels were greater in spleen cells from adults early in infection and then declined, whereas expression levels increased in spleen cells from calves later in the infection process. Together, the data support the concept of 'first come, first serve' cytokine influence over cellular activities, the importance of a type-1 response in the control of an initial infection and the need for tight regulation in order to prevent pathology associated with over production of nitric oxide and inflammatory cytokines.

Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 horses and 415 donkeys) from 6 locations representing different bioclimatic regions were assayed. An analysis of variance, indicated no significant effect of location; however, donkeys were significantly more likely than horses to be seropositive. Management conditions contribute to greater tick infestations and thus Babesia exposure in donkeys than in horses.

Dermacentor hunteri (Acari: Ixodidae): an experimental vector of Anaplasma marginale and A. ovis (Rickettsiales: Anaplasmataceae) to calves and sheep.

The experimental vector competence of laboratory-reared Dermacentor hunteri Bishopp for Anaplasma marginale Theiler and Anaplasma ovis Lestoquard was evaluated by delayed transfer of male ticks from infected to susceptible Holstein calves and from infected to susceptible domestic sheep, respectively. After feeding for 4 or 5 d on rickettsemic acquisition hosts, the ticks were held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 7 or 8 d, then test fed for 5 or 7 d. Additionally, ticks test-fed for 5 d on 2 susceptible calves were removed, held off the host for 7 d, and test-fed for 5 d on a 3rd susceptible calf to test the tick's ability to transmit A. marginale by delayed serial transfer. Tick transmission of A. marginale to 3 test calves and A. ovis to 3 test sheep was demonstrated by blood smear and indirect immunofluorescence serology. These data indicate that males of D. hunteri, a tick commonly found on desert bighorn, Ovis canadensis Shaw, in the southwestern United States and northern Mexico, may be competent natural vectors of these organisms present in desert bighorn populations.

Induction of type 2 cytokines by a staphylococcal enterotoxin superantigen.

Persistent intramammary infections of dairy cows with Staphylococcus aureus may involve immunosuppression mediated by bacterial toxins such as enterotoxins and other super-antigens (SAgs). Previously we found that stimulation of bovine PBMC with staphylococcal enterotoxin C (SEC) induced a unique phenotype of activated CD8+ T cells expressing a newly identified activation molecule, ACT3. In the present study we found that SEC induced the expression of interleukin (IL)-4 and IL-10 mRNAs, two cytokines associated with type 2 responses. Elevated levels of IL-4 and IL-10, observed between day 0 and day 4 of culture, were associated with temporary inhibition of proliferative responses of T cells, evidenced by a decrease in numbers of CD4+ T cells and a small increase in numbers of CD8+ T cells. Vigorous proliferation of T cells occurred between days 4 and 7 of culture and with a bias towards CD8+ T cells. Acquisition of the ACT3+ phenotype by CD8+ T cells was preceded by induction of IL-4 mRNA. Thus, in the bovine system, SAgs may hinder protective responses by inducing type 2 cytokines, which interfere with immune clearance of many microbial pathogens. The results of the study are consistent with the hypothesis that SAgs are involved in immunosuppression, and suggest possible immunomodulatory mechanisms.

Babesia bovis immunity. In vitro and in vivo evidence for IL-10 regulation of IFN-gamma and iNOS.

IL-10 has been shown to have profound immunoregulatory attributes and in the bovine appears to downregulate both Th1- and Th2-like responses. Using RT-PCR, we demonstrate IL-10 in vitro down-regulation of mRNA expression of iNOS, the cytokines involved in nitric oxide signal transduction initiation (IFN-gamma and TNF-alpha), and other mononuclear phagocyte associate cytokines. In addition, using RT-PCR with peripheral blood leukocytes and spleen leukocytes, the Griess reaction, and a killing assay, we provide evidence for the importance of iNOS in a successful immune response to B. bovis infection and for high and persistent IL-10 mRNA expression when the immune response is unsuccessful. We also provide evidence that antibody developed early after an initial infection appears to lack protective attributes (neutralizing and opsonic). Together, the data suggests that IL-10 and IFN-gamma are critical molecules involved in the response to this intraerythrocytic protozoan infection.

Bovine babesiosis. Seroprevalence and ticks associated with cattle from two different regions of Morocco.

A total of 475 bovine sera collected in 1995-1996 from 10 areas belonging to two different bioclimatic strata were tested for antibody activity to Babesia bigemina and Babesia bovis using indirect immunofluorescence (IIF). In the Gharb, the B. bovis seroprevalence was 21.7% and for B. bigemina, 10.8%. The infection rate for either or both species as determined microscopically with Giemsa-stained blood films was 18.9%. The Tiflet area was considered an endemic focus, and the seroprevalence was 42.2% for B. bovis and 40% for B. bigemina. The infection rate by stained blood film microscopy was 66.6%. In the Haouz region, only B. bovis was found, and the seroprevalence was 10.1% with 9.4% microscopically positive blood films. More than 80% of the cattle surveyed were infested by ticks and the mean infestation rate was 36 ticks per animal and 21 ticks per animal in the Gharb and Haouz, respectively. Six species were identified. Hyalomma marginatum, Hyalomma detritum, Hyalomma anatolicum Rhipicephalus bursa, Rhipicephalus sanguineous and Boophilus annulatus. Boophilus annulatus was found in both regions with high prevalence in the Gharb (31.3%). No further correlation was made between the identified species as vectors and the presence of B. bovis and B. bigemina in these areas.

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

The role of IL-10 in iNOS and cytokine mRNA expression during in vitro differentiation of bovine mononuclear phagocytes.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

The distribution of Dermacentor hunteri and Anaplasma sp. in desert bighorn sheep (Ovis canadensis).

The ixodid tick Dermacentor hunteri has been collected intermittently this century, primarily from desert bighorn sheep (Ovis canadensis). Anaplasma spp. are intraerythrocytic rickettsial parasites of ungulates and are vectored in the western United States by ticks of the genus Dermacentor. We tested the hypotheses that D. hunteri would be found infesting all populations of desert bighorn, and that all infested populations would be seropositive for Anaplasma sp. Dermacentor hunteri was found on desert bighorn throughout their range in the Mojave and Sonoran deserts of the southwestern United States and northern Mexico, but not in any portion of the Chihuahuan desert of New Mexico and eastern Arizona or in Baja California Sur, Mexico. Using an indirect immunofluorescence antibody test (IIF), 8 populations of desert bighorn in California with D. hunteri were seropositive for Anaplasma sp. (n = 160). Four populations of desert bighorn with D. hunteri in Arizona (n = 69), 1 in Nevada (n = 22), and I in Utah (n = 14) with D. hunteri were seronegative. Six populations of desert bighorn were uninfested with D. hunteri and were also seronegative. Of these populations, 1 was in California (n = 19), 2 were in New Mexico (n = 33), 2 were in Utah (n = 30), and 1 was in Baja California Sur (n = 14). We found no support for either of our original hypotheses and concluded that both D. hunteri and Anaplasma sp. are limited in their distribution among desert bighorn. We also suggest a cautionary approach to translocations of desert bighorn given the high prevalence of ticks and the unknown effects of Anaplasma sp. on free-ranging bighorn.

Babesia bovis: identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen.

Babesia bovis merozoite proteins presenting as exoantigens in in vitro culture supernatants have been characterized. Bovine antisera to B. bovis exoantigens were used to immunoprecipitate [35S]-methionine metabolically labeled or lactoperoxidase-catalyzed radioiodinated B. bovis merozoite proteins. A total of 24 metabolically labeled proteins ranging in molecular weight from 24,000 to 225,000 Da and 9 radioiodinated proteins with molecular weights varying between 24,000 and 225,000 Da were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies to B. bovis merozoite surface proteins were also used to immunoprecipitate metabolically labeled exoantigens directly from in vitro culture supernatants. These results demonstrate epitopes from at least nine merozoite surface proteins present in the exoantigen fraction, among which are the recently characterized major surface antigens 1 and 2, rhoptry-associated protein 1, and spherical body protein 2.

Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-dependent nitric oxide production.

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.