Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

In vitro transformation of lymphocytes from blood and milk of cows with subclinical paratuberculosis

Lymphocytes from blood or milk of 12 cows were evaluated in vitro for the lymphocyte's capability to proliferate in response to mitogens (phytohemagglutinin-A, concanavalin A, and pokeweed mitogen) and to an antigen prepared from Mycobacterium paratuberculosis (purified protein derivative, PPD-J). Responses of 4 control cows were compared with those of 4 cows subclinically infected with M paratuberculosis and with 4 apparently noninfected herdmates. Blood lymphocytes or milk lymphocytes from control cows had no detectable responses to PPD-J. Blood lymphocytes from infected cows had significant (P less than 0.05) responses to PPD-J, but milk lymphocytes from these cows did not. Conversely, milk lymphocytes from apparently noninfected herdmate cows had significant (P less than 0.05) responses to PPD-J, but blood lymphocytes from these cows did not. There were no significant differences in the responses of blood lymphocytes from control, noninfected, or infected cows to the mitogens. However, milk lymphocytes from infected cows had significantly (P less than 0.05) lower responses than did lymphocytes from the milk of control or noninfected cows to all mitogens. The decreased responsiveness of milk lymphocytes from cows subclinically infected with M paratuberculosis may indicate that immunocompetency of the mammary gland was altered.     

Measurement of lymphoblast proliferative capacity of stimulated blood mononuclear cells from cattle with chronic paratuberculosis.

Concanavalin A (conA) blast proliferation as a quantitative measure of lymphoblast proliferative capacity by blood mononuclear cell supernatants was measured in cattle naturally infected with Mycobacterium paratuberculosis and in healthy control cattle. Blast cell proliferation was significantly reduced in infected animals, compared with control cattle when blood mononuclear cells were stimulated with conA. Proliferation was significantly greater than media control when M bovis purified protein derivative and johnin were used to stimulate cells from the infected group. After sensitizing control and affected cattle with M paratuberculosis bacterin (live M bovis and keyhole limpet hemocyanin in Freund's incomplete adjuvant), infected animals had no difference in blast cell proliferative capacity with the mycobacterial antigens and conA stimulation, whereas healthy animals had significantly increased blast proliferation in response to all the sensitizing antigens. The blast cell proliferative capacity in infected animals with keyhole limpet hemocyanin stimulation was increased significantly after sensitization; however, it remained significantly less than that in the sensitized control group. These data indicate that cattle naturally infected with M paratuberculosis probably produce suboptimal interleukin-2 (IL-2) activity in response to a potent IL-2 inducer (conA) and fail to optimize IL-2 activity when sensitized with a potent immunogen (keyhole limpet hemocyanin).     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

Polymerase chain reaction analysis of TNF-alpha and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis.

Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using the polymerase chain reaction (PCR) and specific bovine oligonucleotide cytokine primers and probes, we examined the expression of messenger RNA for these cytokines in whole blood from M. paratuberculosis infected and uninfected cattle. Cytokine mRNA levels were examined before and after in vitro incubation with E.coli lipopolysaccharide (LPS) and lipoarabinomannan (LAM) purified from M. paratuberculosis. Uninfected calves, experimentally infected calves, and naturally infected cattle all displayed similar cytokine mRNA expression patterns. However, individual animals demonstrated variability in the levels of IL-6 and TNF-alpha mRNA expression as determined by a semiquantitative PCR method using 32P-labelled oligonucleotide probes.     

Spontaneous murine thymocyte comitogenic activity consistent with interleukin-1 in cattle naturally infected with Mycobacterium paratuberculosis

The production of comitogenic activity consistent with interleukin-1 (IL-1) activity by blood monocytes from cattle with naturally acquired paratuberculosis was examined by murine thymocyte proliferation. In addition, IL-1-like activity in response to homologous and heterologous antigens was determined. Activity was determined in nine cattle naturally infected with Mycobacterium paratuberculosis and six non-infected cattle. Comitogenic properties were measured in response to M. paratuberculosis antigen (johnin), bacterial lipopolysaccharide (LPS) as a positive control, and culture media as a negative control. Monocytes from infected cattle spontaneously released high levels of IL-1-like activity in the absence of stimuli and significantly (P less than 0.05) increased activity in response to LPS. With johnin, M. bovis PPD and KLH stimulation, comitogenic activity was similar to spontaneous levels. Non-infected cattle had significantly (P less than 0.05) increased comitogenic activity when blood monocytes were stimulated with KLH, M. bovis PPD, johnin, and lipopolysaccharide when compared with non-stimulated cells in that group. Johnin produced the greatest response in non-infected animals. The data suggest that blood monocytes in infected cattle are non-specifically activated with respect to IL-1 production. Alternatively, a defective regulatory mechanism for IL-1 may be operative in infected cattle. In addition, the previous observation that mycobacterial antigens are potent inducers of IL-1 was also verified.     

Isolation of specific peptides from Mycobacterium paratuberculosis protoplasm and their use in an enzyme-linked immunosorbent assay for the detection of paratuberculosis (Johne's disease) in cattle

An antigen was isolated from the protoplasm of Mycobacterium paratuberculosis by a combination of gel filtration, ion exchange, and affinity chromatography. The purified antigen constituted 7.8% of the total protein in the protoplasm. The specificity and sensitivity of the enzyme-linked immunosorbent assay (ELISA) for paratuberculosis, using the purified antigen, were evaluated with sera from 104 cattle which were examined (surveyed) for M paratuberculosis infection by fecal cultural technique. The ELISA was positive in 50 of 60 infected animals. Five of 44 noninfected animals were also test-positive. When a crude protoplasmic extract was used as antigen in the ELISA, sera from 37 infected and from 18 noninfected animals were test-positive. Cross-reactions were encountered in both complement-fixation test and the ELISA between crude or partially purified M paratuberculosis antigens and antisera to Nocardia asteroides, M avium, M phlei, and M fortuitum. The purified antigen gave no complement-fixation reaction with any of these antisera. In the ELISA, cross-reaction was not found when purified antigen was used and the sera were screened at 1:40 dilution.     

Relationship between Mycobacterium avium subspecies paratuberculosis, IL-1alpha, and TRAF1 in primary bovine monocyte-derived macrophages

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.     

Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.     

Experimental infection of a bovine model with human isolates of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.     

Modulation of cytokine gene expression and secretion during the periparturient period in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression may contribute to the transition from the subclinical to the clinical stage of infection. Understanding the effects of stressors such as parturition on the escalation of disease may provide information that will help to manage JD. The objective of this study was to characterize cytokine gene expression and secretion in periparturient dairy cows naturally infected with MAP. Blood was collected from the jugular vein of healthy noninfected, and subclinically and clinically infected dairy cows for 3 weeks pre-calving to 4 weeks post-calving. Real-time PCR was performed to evaluate the expression of the following cytokine genes by peripheral blood mononuclear cells: IFN-gamma, TNF-alpha, IL-12p35, IL-10, TGF-beta, and IL-4. To assess the effects of parturient immunosuppression on cytokine gene expression, RT-PCR data were analyzed by using 2(-ddCt) values calibrated to dCt value at +1 day relative to calving for each animal. Overall, cytokine gene expression was not influenced by infection status of the cows in this study. However, significant effects in cytokine gene expression were noted across sampling days within the periparturient period. Expression of IFN-gamma by NS and ConA-stimulated PBMCs declined at calving compared with prepartum values in both control and infected cows. Similarly, a decline in expression of IL-4 and IL-10 was observed for cells isolated from subclinically infected cows after stimulation with ConA. ConA-stimulated PBMCs isolated from infected cows secreted higher concentrations of IFN-gamma compared with the controls. A significant decline in IFN-gamma secretion was noted for MPS-stimulated cells for clinical cows from -21 days to +1 day. Stimulating cells with MPS resulted in greater secretion of IL-10 by infected cows during the postpartum period. A trend was also observed for higher TGF-beta secretion by NS PBMCs isolated from clinical cows in the postpartum period. Cells isolated from clinically infected cows and stimulated with MPS secreted higher levels of nitric oxide throughout the periparturient period when compared to control or subclinically infected cows. These data suggest that parturition is a very dynamic time period for host immunity, with potential for altered immunity to hinder the ability of dairy cows to thwart infectious diseases.     

Analysis of the effect of inapparent bovine paratuberculosis.

Inapparent paratuberculosis was studied in a Guernsey herd with a history of paratuberculosis (Johne's disease); the herd was maintained at between 900 and 950 cattle. Fecal specimens and intestinal tissue specimens from any of these that were slaughtered were examined culturally for the presence of Mycobacterium paratuberculosis. The reasons given for culling and slaughtering of cows from this herd were compared with infection status determined by culturing. Less than one-third of the culturally confirmed paratuberculous cattle were culled because of clinically apparent paratuberculosis. Mastitis was the reason for culling 3.6% of the non-infected cattle and 22.6% of the cattle with inapparent paratuberculosis. Infertility was also significantly higher in cows with inapparent paratuberculosis than in noninfected cows in the same herd. Separation of parturient dams and calves from the rest of the herd was shown to materially reduce the level of infection and incidence of clinical paratuberculosis.     

Serodiagnosis of bovine paratuberculosis by use of a dot enzyme-linked immunosorbent assay

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.     

Mucosal immune response in cattle with subclinical Johne's disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of wild and domestic ruminants. During a long subclinical period, the organism persists in the intestine despite systemic cellular and humoral immune responses. To explore the mucosal immune response in Johne's disease, we isolated mononuclear leukocytes from the ileum of cows naturally infected with M. avium subsp. paratuberculosis and from cows that were not infected. We evaluated the immunophenotype of these cells and the proliferative responses after the addition of M. avium subsp. paratuberculosis sonicate or B-cell or T-cell mitogens. Although the percentage of T cells was increased in infected cows, these cells consisted mostly of memory (CD2+CD62L-) and regulatory (CD4+CD25+) T cells. Further evidence of immune hyporesponsiveness included a decrease in the percentage of T cells with an activated phenotype and a decrease in cells expressing major histocompatibility factor class II (MHC class II). Unlike the spleen, ileal lymphocytes from infected cows failed to proliferate in response to M. avium subsp. paratuberculosis sonicate. Additionally, ileal lymphocytes from infected cows proliferated poorly in response to concanavalin A and pokeweed mitogen, suggesting generalized T cell and B cell hyporesponsiveness. These results indicate that a state of tolerance may exist in the intestine of cows subclinically infected with M. avium subsp. paratuberculosis organisms in subclinically infected cows. This effect may be induced, at least in part, by proliferation of regulatory T cells that nonspecifically suppress mucosal immune responsiveness.     

Nested PCR on blood and milk for the detection of Mycobacterium avium subsp paratuberculosis DNA in clinical and subclinical bovine paratuberculosis

OBJECTIVE: To determine the potential of PCR on blood and milk to detect cattle infected with Mycobacterium avium subsp paratuberculosis. PROCEDURE: A nested PCR method probing for IS900 was developed and compared to ELISA serology in 11 clinically infected and 46 subclinically infected, lactating Holstein cows from a herd with confirmed paratuberculosis (Johne's disease). RESULTS: When compared to serum ELISA the nested blood- and milk PCRs were equal in identifying DNA from clinically infected animals. The PCR procedures also gave positive DNA results with some subclinically infected animals when these only gave suspicious or negative results in the ELISA test. Most clinically and subclinically infected animals were detected with milk PCR. CONCLUSION: Since there may well be a haematological phase in paratuberculosis, nested PCR testing of blood and milk samples shows potential to detect animals subclinically infected with M a paratuberculosis. More subclinically infected animals need to be tested and confirmed infected before estimates of sensitivity and specificity can be made.     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

Cytokine gene expression profiles in peripheral blood mononuclear cells from Neospora caninum naturally infected dams throughout gestation

Neospora caninum is a major cause of abortion in cattle but it is not known why some infected animals suffer abortion while others do not. An essential role in protective immunity against N. caninum has been proposed for Th1 cytokines such as IFN-γ and IL-12 although cytokine patterns in N. caninum infected pregnant cattle have been scarcely addressed. In this study, gene expression of the cytokines IFN-γ, IL-12, IL-10, IL-4 and TNF-α was analyzed by real time RT-PCR in peripheral blood mononuclear cells in N. caninum naturally infected dams throughout pregnancy. Blood samples were drawn from 18 cows (13 N. caninum seropositive and 5 N. caninum seronegative) on Days 45, 90, 120, 150, 180 and 210 of pregnancy or until abortion. Four seropositive animals aborted. Compared to the seronegative animals, N. caninum infected dams showed up-regulated mRNA levels of the Th1 cytokines, IFN-γ, TNF-α and IL-12p40, along with up-regulation of the T regulatory (Treg) cytokine IL-10. In contrast, expression levels of IL-4 (Th2 cytokine) did not differ significantly among the different groups throughout the study period. Our findings indicate clear differences in peripheral blood cytokine gene expression levels during pregnancy between animals naturally infected with N. caninum and seronegative control animals. To the best of our knowledge, this is the first study to examine the gene expression of Th1, Th2 and regulatory cytokines in the peripheral blood of pregnant cows naturally infected with N. caninum.