Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle

Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE₂) and luteolytic (PGF₂) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF₂, LH, PGE₂, LH + PGE₂ or LH + PGF₂. Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF₂ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF₂ a tendency to reduce mitogenesis. Thus, prostaglandin F₂ may play a role on vascular regression of the CL during the late luteal phase in the mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

绵羊发情周期子宫内膜微血管密度与VEGF的表达关系

以绵羊发情周期的子宫内膜为研究对象,采用免疫组化方法定量检测绵羊发情周期子宫内膜的微血管密度（MVD）和血管内皮生长因子（VEGF）表达量。结果表明：MVD的标记物CD34和VEGF在绵羊发情周期的子宫内膜中呈现相同的表达特征,即表达位点均在子宫内膜上皮固有层及肌层;两者均在发情后0d开始表达,5d最高。5d开始到15d表达量缓慢下降。子宫内膜VEGF表达量和MVD相关系数r=0.669,P=0,表明子宫角中VEGF表达量和MVD显著正相关。     

Cytoplasmic estrogen and progesterone receptors in canine endometrium during the estrous cycle

Estradiol and progesterone receptors (ER, PR) were characterized and measured in cytosols from canine endometrium, using saturation and sucrose-gradient centrifugation radioassays. Both receptors were demonstrated to be steroid- and tissue-specific saturable proteins, which bound the respective steroids with high affinity (dissociation constant [Kd] approximately 10(-9)M). Serum estradiol, progesterone, and endometrial cytosol receptor concentrations and receptor-binding affinity were measured for 25 bitches from which samples were obtained at 5 stages of the estrous cycle (5 bitches each): anestrus (A), the 3rd day of proestrus (P3), the 3rd day of estrus (E3), the 12th day after onset of estrus (E12), and the 28th day after onset of estrus (E28). Mean (+/- SEM) serum estradiol concentrations were 17.0 +/- 2.2 (A), 55.4 +/- 5.0 (P3), 89.4 +/- 24.9 (E3), 41.0 +/- 5.9 (E12), and 50.6 +/- 3.9 (E28) pg/ml. Mean (+/- SEM) serum progesterone concentrations were 0.4 +/- 0.1 (A), 1.5 +/- 0.2 (P3), 17.3 +/- 7.5 (E3), 41.6 +/- 9.5 (E12), and 25.8 +/- 3.2 (E28) ng/ml. Concentrations of ER increased significantly from 1.06 pmol/g of uterus during stage A to a peak concentration of 6.18 pmol/g of uterus at E12, followed by a gradual decrease to 0.69 pmol/g of uterus by E28. The PR concentrations increased from 3.01 pmol/g of uterus in stage A to 17.32 pmol/g of uterus at P3; PR concentrations, thereafter, decreased gradually to 1.85 pmol/g of uterus by E28. Dissociation constants were significantly higher at E12 for the ER (Kd = 2.6645 X 10(-9)M) and at P3 for the PR (Kd = 5.8282 X 10(-9)M) than at the other stages examined, indicating a decrease in receptor affinity during the periods of high receptor concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.     

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

L-硝基精氨酸对阿尔茨海默症小鼠脑海马nNOS阳性神经元分布、NOS活性及NO含量变化的影响

为了研究D-半乳糖联合铝诱导的小鼠阿尔茨海默症(AD)脑海马一氧化氮合酶(NOS)活性和一氧化氮(NO)含量的变化及L-NNA和盐酸多奈哌齐对其变化的影响,探讨NO在阿尔茨海默症中的作用机制及2种药物对脑神经元的保护作用。选取2月龄健康昆明小鼠160只,体质量(20±2)g,随机分为正常对照组、模型组、盐酸多奈哌齐治疗组及L-NNA治疗组,利用D-半乳糖联合三氯化铝建立小鼠AD模型,应用生化检测技术测定各组脑海马在造模后每周NOS活性及NO含量。结果表明,模型组海马内NOS活性开始呈缓慢升高,从第4周开始呈显著升高,保持较高含量至12w造模结束;两治疗组脑海马NOS活性在各时间点极显著或显著低于模型组(P0.01或P0.05),并且L-NNA在4~8周时降低脑海马NOS活性的程度好于盐酸多奈哌齐治疗组(P0.05);NO含量的变化随着NOS活性的变化而变化;利用SABC免疫组织化学方法检测各组脑海马神经型NOS(nNOS)阳性神经元,发现模型组脑海马nNOS阳性神经元密度降低,细胞着色变淡,胞体截面积和最长突起长度变小,经过治疗后神经元密度增加,胞体截面积和最长突起长度显著改善(P0.05)。结果提示NO参与了AD形成过程,高浓度的NO能发挥神经毒性作用损害脑组织;L-NNA通过抑制NOS的活性,降低了脑海马NO的含量,对阿尔茨海默症中海马神经元具有明显的保护作用。     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.     

Endometrial gland surface density and hyperaemia of the endometrium during early pregnancy in the mare

REASON FOR PERFORMING STUDY: The morphology and surface density of endometrial glands responsible for the nutrition of the placenta requires further study employing computer assisted morphometric analysis. OBJECTIVES: To analyse changes in the morphology and surface density of endometrial glands situated adjacent to, and remote from, the conceptus during the first 30 days of gestation. METHODS: Endometrial biopsies were recovered, under videoendoscopic control, from both nonpregnant and pregnant mares 12 days after ovulation, as well as from pregnant mares on Days 16, 20, 25 and 30 of gestation. One biopsy was taken at the base of the nongravid uterine horn and the other from beneath the conceptus at the base of the gravid horn. The samples were evaluated by computer assisted morphometric analysis to determine the surface area per unit volume (Sv) of endometrial glands in the stratum spongiosum. RESULTS: No differences in morphology or density of the glands were discernable between pregnant and nonpregnant mares on Day 12 day after ovulation. On Day 16, however, the glands beneath the conceptus appeared to be at a significantly lower density than the clearly more tortuous glands at the base of the nongravid horn. Between Days 20 and 30 of gestation the endometrium in direct contact with the conceptus was visibly more reddened and hyperaemic than elsewhere in the uterus. However, the Sv of glands beneath the conceptus had now increased again so they appeared similar to those in the nongravid horn, due mainly to a reduction in the level of stromal oedema in the former. CONCLUSIONS AND POTENTIAL RELEVANCE: The embryo-maternal interactions that are vital during early pregnancy to suppress the cyclical luteolytic cascade and support nidation of the conceptus, were characterised here with local endometrial hyperaemia, histotroph production and a decrease in endometrial gland Sv at the commencement of the stationary phase. Both the components secreted by the endometrial glands and the factors liberated by the conceptus during early pregnancy need further investigation.     

Decreased plasma concentration of nitric oxide metabolites in dogs with untreated mitral regurgitation

Endothelium-dependent (nitric oxide [NO]-mediated) vasodilation is impaired in humans with heart failure. This dysfunction is an important therapeutic target. The plasma concentration of the NO metabolites nitrate and nitrite (collectively referred to as NOx) is a measure of whole-body NO production, provided that the dietary intake of the ions is low. Fifty clinically healthy dogs older than I year (median 5.0 years; interquartile interval 2.6-8.2 years) were studied, including 9 controls of various breeds, 23 Cavalier King Charles Spaniels (CKCSs) with no or minimal mitral regurgitation (MR), 9 CKCSs with mild MR (regurgitant jet occupying 15-50% of the left atrial area), and 9 CKCS with moderate to severe MR (jet >50%) due to myxomatous valve disease. None of the dogs received medication. The dogs were given NOx-free water and a diet with a low concentration of NOx for 96 hours before blood sampling. Multiple linear regression analysis revealed that dog group, but not gender, age, serum creatinine concentration, and platelet count, was associated with NOx concentrations. Control dogs had the same NOx concentration (median 20.0 microM; interquartile interval 15.1-25.5 microM) as CKCSs without MR (median 18.7 microM; interquartile interval 15.5-25.9 microM). Compared to CKCSs without MR, the NOx concentration was lower in CKCSs with mild (median 12.9 microM; interquartile interval 11.0-13.5 microM; P = .04) and moderate to severe (median 11.2 microM; interquartile interval 6.9-17.1 microM; P = .02) MR. In conclusion, CKCSs with mild to severe, clinically silent MR have decreased plasma NOx concentrations, suggesting that endothelial dysfunction develops early in the course of developing MR in dogs.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats

Nitric oxide (NO) has previously been shown to be responsible for nitrogen mustard (NM)-induced tissue toxicity. Excessive amounts of NO are known to be able to produce peroxynitrite, an important reactive nitrogen compound, by reacting with superoxide. Previous studies reported that NO synthase inhibitors are able to prevent NM toxicity. The aim of this study was to evaluate whether peroxynitrite is also responsible for NM-induced lung tissue damage in rats. Wistar rats were divided into four groups. NM was injected intratracheally and was treated with the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG) (intraperitoneal) or the peroxynitrite scavenger ebselen (intragastric). Control animals were exposed to saline only. NM injection caused both oxidative and nitrosative stress, reflected by dramatically increased levels of the lipid peroxidation end product malondialdehyde (MDA), iNOS activation and urine nitrite-nitrate (NOx) values. Histopathological evaluation demonstrated lung damage with NM exposure. AG blocked iNOS activation and decreased urine NOx levels, and resulted in less histopathological changes in the lung. Although the histopathological outcome was found to be similar to AG, ebselen did not change urinary NOx or lung iNOS levels. Furthermore, ebselen was more able than AG to protect against MDA accumulation. In conclusion, the ability of ebselen to prevent against lung damage without blocking NO synthesis suggests that peroxynitrites may have an important role in the pathogenesis of NM toxicity in addition to NO.     

Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk

The aim of this study was to investigate total antioxidant (TAC), and oxidant capacity (TOC) and nitric oxide (NO) levels in milk of cows with subclinical mastitis. Brown Swiss and Holstein breed cows were screened with California Mastitis Test (CMT) to determine mammary glands with subclinical mastitis. Moreover, somatic cell counts (SCC) were determined electronically in all milk samples. Mammary quarters were classified as healthy (n = 25) or subclinical mastitis (n = 35) based on CMT scores and somatic cell count (SCC: ?200,000/ml or >200,000/ml) in milk. Nitric oxide, TOC and SCC levels were significantly higher (p < 0.001, p < 0.005 and p < 0.001, respectively) in milk from mammary quarters with subclinical mastitis compared to those from healthy mammary quarters. In conclusion, subclinical mastitis results in higher NO concentrations, TOC and SCC, and NO and TOC were positively correlated with SCC. Moreover, alterations in NO levels and TOC in milk could be used as an alternative diagnostic tool to screen for subclinical mastitis.     

Luteotrophic and luteolytic effects of nitric oxide in sheep are dose-dependent in vivo

It has been suggested that nitric oxide (NO) acts in either an anti-luteolytic or in a luteolytic manner, but the mechanism for these opposing roles is unclear. We hypothesized that NO may act in a dose-dependent manner to regulate luteal function, whereby low concentrations of NO might stimulate luteal progesterone production (i.e. luteotrophic) and high concentrations of NO might reduce concentrations of plasma progesterone (i.e. luteolytic). To test this hypothesis we infused increasing concentrations of the fast-acting NO donor, dipropylenetriamine NONOate (DPTA), into the arterial supply of sheep with ovarian transplants bearing a corpus luteum (CL). Infusions were performed on sheep with CL 11 days of age (n=9) or over 30 days of age (n=15). We measured changes in the concentration of progesterone in ovarian venous plasma during the 1-h infusion and for 24h after the infusion, and then compared the mean concentration of progesterone between treatment groups for effects by dose and dose by period interactions. Compared with saline-treated controls (n=6), the highest dose of 1000 microg/min DPTA (n=6) reduced (P0.05) in sheep infused with the lowest dose of 1 microg/min DPTA (n=6) compared with controls. We conclude that NO regulates luteal function in a dose-dependent manner in sheep in vivo.     

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

Expression profiles of perforin,granzyme B and granulysin genes during the estrous cycle and gestation in the bovine endometrium

The conceptus is susceptible to destruction by maternal cytotoxic lymphocytes, which have cytotoxic potential. Therefore, it is expected that mechanisms for regulating cytotoxic lymphocytes exist, but little is known about the expression of cytotoxic genes in the endometrium. In the present study, we examined the spatial and temporal expression patterns of the cytotoxic genes perforin, granzyme B, and granulysin during the estrous cycle and gestation in the bovine endometrium. Endometrial tissues were collected from cows during the estrous cycle and gestation. The gene expression patterns of the three cytotoxic genes were examined using quantitative polymerase chain reaction and in situ hybridization, and cytotoxic lymphocyte subsets were characterized using immunohistochemistry. During mid‐ to late gestation in the intercaruncular (ICAR), granulysin expression was significantly increased, and a large number of granulysin‐expressing cells were localized in the luminal epithelium. Perforin and granzyme B displayed similar expression profiles and were highly expressed in the peri‐implantation endometrium, but few cells expressing these genes were found in the endometrial stroma. In conclusion, these findings suggest that in the ICAR epithelium granulysin may play important roles in the establishment and maintenance of gestation during normal pregnancy.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.