Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes.

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.     

Leishmune vaccine: the newest tool for prevention and control of canine visceral leishmaniosis and its potential as a transmission-blocking vaccine

Canine visceral leishmaniosis is a life-threatening disease caused by Leishmania infantum. For quite some time, specialists in leishmaniosis have tried to develop more affordable and effective control measures against this disease. In this search, the first vaccine against canine visceral leishmaniosis was recently licensed in Brazil. In the light of recent research, the Leishmune vaccine might be seen as the newest tool for prevention and control of canine visceral leishmaniosis. Moreover, the potential of the Leishmune as a transmission-blocking vaccine has recently been demonstrated, indicating its usefulness in the control of zoonotic visceral leishmaniosis.     

Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis.After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role.The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis.Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.     

Differential serological testing by simultaneous indirect immunofluorescent antibody test in canine leishmaniosis and ehrlichiosis

A mixed indirect fluorescence antibody test (IFAT), based on cultured promastigotes Leishmania infantum and formol-inactivated suspension of cells infected with the bacteria Ehrlichia canis, was applied to make a differential diagnosis between canine ehrlichiosis and leishmaniosis. A titre greater than 80 was considered positive for antibodies to E. canis and suggestive of antibodies to L. infantum. Positive sera were titrated subsequently by serial dilutions to confirm antibodies positive to Leishmania and establishing the antibody titre of both pathogens. Fluorescence was absent with negative control sera and background staining was minimal. No serological cross-reactions between positive sera for L. infantum or E. canis were detected. Results obtained by mixed IFAT did not differ when the same serum IFAT standard was compared. The test showed equivalent sensitivity (100%). The specifities were 100% for L. infantum and 98.5% for E. canis. The equivalence in sensitivity was confirmed by calculating the correlation coefficient between IFAT standards and mixed IFAT (r>or=0.99 for both pathogens). The results of our investigations demonstrated that mixed IFAT is a specific means of establishing serological differential diagnosis of canine leishmaniosis and ehrlichiosis.     

Detection of Leishmania infantum in captive wolves from Southwestern Europe

The aim of the present study was to determine the prevalence of Leishmania infantum infection in a wild reservoir host (Canis lupus) throughout an endemic area for the disease (Southern Europe). For that reason, the serum and peripheral blood samples of 33 captive wolves from the European Breeding of Endangered Species Programme (EEP) were analyzed using the enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qPCR). L. infantum was detected in three samples from Central Portugal and Central and Northern Spain. Even though L. infantum infection in positive samples was low, surveillance of zoonotic leishmaniosis in this population is recommended as the parasite load could be higher in other tissues due to parasite tropism and most of the EEP institutions studied are located in endemic areas for canine leishmaniosis in Europe.     

Immune response to Leishmania infantum in healthy horses in Spain

Leishmania infantum infection has recently been described in horses in Europe. We report the results of a study on the immune response to L. infantum in horses living in an area endemic for leishmaniosis in NE Spain. Two ELISAs using protein A and anti-horse IgG conjugates were adapted to measure specific antibodies to L. infantum in horse sera. A lymphocyte proliferation assay (LPA) of peripheral blood mononuclear cells to L. infantum antigen was also performed to detect specific cellular immune response to Leishmania. Anti-L. infantum antibodies were detected in the serum of 16 of the horses studied (n=112) using the protein A assay but not in the assay using the anti-horse IgG conjugate. Specific lymphocyte proliferation was observed in 20 out of 55 horses. This study shows that horses in the area studied mount specific immune responses to L. infantum, and must therefore be considered among the species exposed to the parasite in this region. The infrequency of leishmaniosis in horses suggests that the immune response in this species is effective in controlling the infection.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain

Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.     

Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.     

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.     

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.     

The importance of canine leishmaniosis in non-endemic areas, with special emphasis on the situation in Germany

This review article summarizes the situation of canine leishmaniosis in Germany. Published case studies on infections with Leishmania (L.) infantum in either humans or dogs are analyzed. Diagnosed cases of infections by Leishmania spp. in humans and animals are not a notifiable disease in Germany or other European countries. Taking this into consideration one may assume that there might be a significant gap between the analyzed and reported cases and the infectious status within the country. The reported case studies and results from surveys indicate that the majority of all L. infantum infections are acquired during travelling in endemic regions, predominantly the Mediterranean region. However there are cases reported from human infections and growing number of cases in dogs, where the case history may indicate an autochthonous infection within Germany, a country within a non-endemic region. The current data from entomological field studies proved the presence of two phlebotomine sand fly species. Phlebotomus (P.) mascittii, an anthropophilic sand fly species and P. perniciosus a proven vector of L. infantum. The impact from a growing leishmania-positive dog population within Germany, the distribution of at least two sand fly species, one with vector potential in the light of climate change and other non-vectorial transmissions are summarized.     

Flow cytometric analysis of cellular immune responses in dogs experimentally infected with a North American isolate of Leishmania infantum

Canine leishmaniasis caused by Leishmania infantum is endemic in the foxhound population in North America. Studies of canine leishmaniasis in the Mediterranean basin indicate a role for both CD4+ and CD8+ lymphocytes with clinical illness and in asymptomatic dogs. Limited information is available on the strain of L. infantum infecting foxhounds in North America. The present study investigated changes in cellular immune responses in dogs experimentally infected with 1x10(7) (low dose, LD; N=4) or 2x10(8) (high dose, HD; N=4) promastigotes of a United States isolate of L. infantum and control dogs (N=2) for 72 weeks. Density gradient separation was used to enrich for peripheral blood lymphocytes from canine blood. Lymphocyte subsets (CD4+ and CD8+) were quantified by flow cytometric analysis. Lymphocyte population expression levels over the course of the present study were compared to clinical status of the dog and antibody responses in infected and control dogs. No significant differences (P>0.05) were observed in either CD4+ or CD8+ lymphocyte expression in of the groups over the experimental period. This study suggests that the cellular immune responses to North American L. infantum in experimentally infected dogs may differ from other strains of L. infantum.     

Interaction of antigen presenting cells with mycobacteria

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNF and IL-10 whereas macrophages produced TNF, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.