Therapeutic effect of anti-TNF-alpha antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-alpha antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-alpha antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-alpha antibody and LVFX. Anti-TNF-alpha antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Effects of GnRH in combination with PGF2α on the dynamics of follicular and luteal cells in post-pubertal Holstein heifers

Holstein heifers were randomly allotted by weight, age and body condition score to one of three treatments to test the hypothesis that GnRH administration concurrent with PGF_2α injection would advance follicle or corpus luteum (CL) development parallel to an induced luteolysis of the pre-existing CL. Heifers in the control group (n = 14) received two treatments of PGF_2α(25 mg, im) given 10 days apart. Groups 2 (n = 14) and 3 (n = 14) received an additional treatment of GnRH (100 μg, im) after the first and second PGF_2α respectively. Estrus detection began immediately after PGF_2α and continued for 80 h. Blood sampling was initiated 7 days prior to the first PGF_2α (day − 7) and continued on days 0, 7, 10 (prior to the second PGF_2α), 17 and 24. Heifers were artificially inseminated after the second PGF_2α and pregnancy diagnosed at 60 days. There was a trend (P < .10) toward a lower estrus response in group 3 when compared to the other groups. Pregnant heifers in group 2 had lower progesterone (0.44 ± 0.09 vs. 1.72 ± 0.56 ng/ml) a week after the second PGF_2α than the non-pregnant animals in that group (P < .05). Similar results were observed in the control group but only within the responding heifers (0.61 ± 0.08 vs. 0.93 ± 0.03 ng/ml; P < .05). Progesterone in heifers in group 2 remained high on day 0, 7, and 10 (1.48 ± 0.37, 1.23 ± 0.39, 1.96 ± 0.36 ng/ml) in spite of the treatment with PGF_2α. This data suggest that administration of GnRH following PGF_2α alters bovine luteal and/or follicular cell function.     

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioides brasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-γ production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-γ. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-γ and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.     

Some properties of beta toxin produced by Clostridium haemolyticum strain IRP-135

Six culture media were evaluated for the optimization of β-toxin production by Clostridium haemolyticum (strain IRP-135) using both batch and dialysis culture techniques. The lethal component of β-toxin remained active for 13 days when maintained at 37°C but was inactivated by heating at 60° for 20 min. A 1 : 10,000 dilution of trypsin inactivated the toxin in 15 min. Preliminary data from electrophoresis in SDS acrylamide gel indicate the molecular weight of the β-toxin to be approximately 32,000.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Initial adherence of EPEC,EHEC and VTEC to host cells

Initial adherence to host cells is the first step of the infection of enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC) and verotoxigenic Escherichia coli (VTEC) strains. The importance of this step in the infection resides in the fact that (1) adherence is the first contact between bacteria and intestinal cells without which the other steps cannot occur and (2) adherence is the basis of host specificity for a lot of pathogens. This review describes the initial adhesins of the EPEC, EHEC and VTEC strains. During the last few years, several new adhesins and putative colonisation factors have been described, especially in EHEC strains. Only a few adhesins (BfpA, AF/R1, AF/R2, Ral, F18 adhesins) appear to be host and pathotype specific. The others are found in more than one species and/or pathotype (EPEC, EHEC, VTEC). Initial adherence of EPEC, EHEC and VTEC strains to host cells is probably mediated by multiple mechanisms.     

Duration of feeding linseed diet influences peroxisome proliferator-activated receptor γ and tumor necrosis factor gene expression, and muscle mass of growing–finishing barrows

The aim of the study was to investigate the effect of duration of feeding linseed (rich in n-3 PUFA) on peroxisome proliferator-activated receptor γ (PPARγ) and tumor necrosis factor (TNF-α) gene expression, and muscle mass of growing–finishing barrows. Two isoenergetic and isonitrogenous diets were formulated, and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%. Twenty-four Landrace × Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to four treatments with six individuals per treatment. Pigs in treatment 1 (T1) fed the control diet throughout the experimental period, while pigs in T2, T3 and T4 fed the control diet except for 30, 60, and 90 d prior to slaughter when the linseed diet were fed. The experiment was conducted for 90 days. The longissimus muscle mass and each muscle mass in the hind leg were weighted. PPARγ and TNF-α mRNA expression levels in muscle, spleen and adipose tissue, and plasma concentrations of TNF-α data were measured and analyzed. The results showed that the longissimus muscle mass, quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly (P < 0.01) as prolonged the time of feeding linseed diet. The expression of PPARγ in longissimus muscle and spleen increased (P < 0.01) linearly as prolonged the time of feeding linseed diet, while the expression of PPARγ in adipose tissue were not affected (P = 0.095). Duration of linseed addition linearly decreased (P < 0.01) TNF-α gene expression levels in the longissimus dorsi muscle, adipose and spleen, and serum concentration of TNF-α as well. The expression levels of PPARγ negatively correlated with the expression of TNF-α in muscle (R² = 0.70, P < 0.001) and spleen (R²R² = 0.77, P < 0.001) respectively. Likewise, PPARγ expression level in spleen (R²R² = 0.59, P < 0.01) or muscle (R²R² = 0.52, P < 0.05) negative correlated with serum TNF-α concentration, while there were significant quadratic relation between muscular PPARγ (R²R² = 0.80, P < 0.01) or muscular TNF-α (R²R² = 0.87, P < 0.01) expression and the longissimus dorsi muscle mass. These data suggested that duration of feeding linseed diet lead to a linear decrease of TNF-α gene expression, which may increase the muscle mass in growing–finishing barrows, at least in part, through a PPARγ-dependent mechanism.     

Vaccination of cows with rough Escherichia coli mutants fails to protect against experimental intramammary bacterial challenge

Vaccination of cows with rough Escherichia coli mutants fails to protect against experimental intramammary bacterial challenge. Vaccine A, a heat-killed Re mutant of strain K12, (UB 1574), was administered as a single parenteral and local dose to 5 cows with 3 control animals and Vaccine B, a heat-killed mutant of O111:B4 (J5) was administered as three parenteral doses into 5 cows with 5 control animals. Following intramammary challenge with a smooth wild-type strain (P4), an acute, severe clinical mastitis developed in all 14 quarters (9 vaccine A and 5 vaccine B) of the vaccinated animals which was indistinguishable from that in the 11 quarters of the control animals. Following vaccine B there was an elevation in serum IgG1 and IgG2 antibody to the common core antigen of endotoxin which, in contrast to the control animals, showed a further increase after intramammary infection.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Characterization of Shiga toxin – producing Escherichia coli infections in beef feeder calves and the effectiveness of a prebiotic in alleviating Shiga toxin - producing Escherichia coli infections

Background

In the less-sensitive mouse model, Shiga toxin-producing Escherichia coli (STEC) challenges result in shedding that reflect the amount of infection and the expression of virulence factors such as Shiga toxins (Stx). The purpose of this study was to characterize the contribution of STEC diversity and Stx expression to shedding in beef feeder calves and to evaluate the effectiveness of a prebiotic, Celmanax®, to alleviate STEC shedding. Fecal samples were collected from calves at entry and after 35 days in the feedlot in spring and summer. STECs were evaluated using selective media, biochemical profile, serotyping and Stx detection. Statistical analysis was performed using repeated measures ANOVA and logistic regression.

Results

At entry, non-O157 STEC were dominant in shedding calves. In spring, 21%, 14% and 14% of calves acquired O157, non-O157 and mixed STEC infections, respectively. In contrast, 45%, 48% and 46% of calves in summer acquired O157, non-O157 and mixed STEC infections, respectively. Treatment with a prebiotic, Celmanax®, in spring significantly reduced 50% of the O157 STEC infections, 50% of the non-O157 STEC infections and 36% of the STEC co-infections (P = 0.037). In summer, there was no significant effect of the prebiotic on STEC infections. The amount of shedding at entry was significantly related to the number and type of STECs present and Stx expression (r² = 0.82). The same relationship was found for shedding at day 35 (r² = 0.85), but it was also related to the number and type of STECs present at entry. Stx - producing STEC infections resulted in 100 to 1000 × higher shedding in calves compared with Stx-negative STECs.

Conclusions

STEC infections in beef feeder calves reflect the number and type of STECs involved in the infection and STEC expression of Stx. Application of Celmanax® reduced O157 and non-O157 STEC shedding by calves but further research is required to determine appropriate dosages to manage STEC infections.     

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.     

川西北牦牛肉中产志贺毒素大肠杆菌的分离鉴定及stx2基因的序列分析

为了解中国川西北牦牛肉中产志贺毒素大肠杆菌(STEC)的携带情况及stx2的亚型和特征,试验将采集的204份川西北牦牛肉样品(各25g)增菌培养后,每份挑取5个可疑菌落,采用stx1、stx2双重PCR方法检测STEC,对分离株中的stx2分型并克隆测定stx2编码区序列。结果显示,在204份样品中分离出8株STEC,平均分离率为3.9%(8/204);存在4个不同的O血清型,分别为O38(4)、O50(1)、O74(2)、O150(1);在6株含有stx2的菌株中,其中有2株为stx2a型、4株为stx2c型。结果表明牦牛源分离株氨基酸序列与人源和牛源菌株同源性较高;由stx2A、B亚基的氨基酸序列系统进化树可知,牦牛源分离株与人源、牛源菌株聚为一支,表明它们之间遗传距离相对较近,牦牛源stx2各自分布在自己的小分支中,表明牦牛源STEC stx2与人源和牛源stx2相比,尽管亲缘关系较近,但仍存在一定程度的差异。     

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.     

Endotoxin in the conscious piglet: Its effects on some general and gastrointestinal myoelectrical parameters

The effect of an intravenous bolus injection of endotoxin, 0.1, 1 or 10 g/kg, on rectal temperature, clinical appearance, haematological parameters, and on gastrointestinal electrical activity was examined in 11 conscious piglets of 4–5 weeks of age, with implanted electrodes in the antrum pylori, duodenum, jejunum and ileum. All doses resulted in a significant and dose-dependent increase in rectal temperature, in pronounced clinical signs and in distinct changes in haematological values. These included shivering, depression, respiratory distress, a leukopenia (0.1 g/kg) or a leukocytosis (1 g/kg) with a shift to the left, an accelerated sedimentation rate and a decreased packed cell volume. Doses of 1 and 10 g/kg induced a transient inhibition of gastroduodenal electrical activity. These results suggest that, in the piglet, endotoxin primarily manifests general clinical signs and that the gastrointestinal effects coincide with these.     

Cytokine profiles, apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P < 0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.     

Immunostimulatory effects of natural human interferon-alpha (huIFN-α) on carps Cyprinus carpio L.

Human interferon-α (huIFN-α) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-α in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-α were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1β, tumor necrosis factor-α and interleukin 10. Low doses of huIFN-α were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-α significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-α-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-α on the carp immune system and highlights the immunomodulatory role of huIFN-α in fish.     

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities.

Results

Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45–71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Conclusion

The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.     

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.