No abstract

-Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/µL) and 146 thrombocytopenic samples (less than 200 000/µL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/µL (Group B) and 84 samples with less than 100 000 platelets/µL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis. Ehrlichia canis / thrombocytopenia / platelet counts / screening / PCR

Propolis is a bee product and its immunomodulatory action has been the subject of intense investigation lately. The recent discovery and characterization of the family of Toll-like receptors (TLR) have triggered a great deal of interest in the field of innate immunity due to their crucial role in microbial recognition and development of the adaptive immune response. This work aimed to evaluate propolis's effect on TLR-2 and TLR-4 expression and on the production of pro-inflammatory cytokines (IL-1beta and IL-6). Male BALB/c mice were treated with propolis (200 mg/kg) for three consecutive days, and TLR-2 and TLR-4 expression as well as IL-1beta and IL-6 production were assessed in peritoneal macrophages and spleen cells. Basal IL-1beta production and TLR-2 and TLR-4 expression were increased in peritoneal macrophages of propolis-treated mice. TLR-2 and TLR-4 expression and IL-1beta and IL-6 production were also upregulated in the spleen cells of propolis-treated mice. One may conclude that propolis activated the initial steps of the immune response by upregulating TLRs expression and the production of pro-inflammatory cytokines in mice, modulating the mechanisms of the innate immunity.

Preeclampsia (PE) is a specific syndrome of pregnancy, characterized by hypertension and proteinuria. This pathology is associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals may activate an intracellular complex called inflammasome, which is important for processing and release of inflammatory cytokines. This study investigated the state of monocyte activation, both endogenous and stimulated with monosodium urate (MSU), by gene expression of NLRP1 and NLRP3 receptors as well as their association with inflammatory cytokines expression. Monocytes were obtained from peripheral blood of 23 preeclamptic pregnant women, 23 normotensive pregnant women (NT) and 23 healthy non-pregnant women (NP). Inflammasome activation was evaluated by the gene expression of NLRP1, NLRP3, caspase-1, IL-1β, IL-18 and TNF-α by RT-qPCR in unstimulated monocytes (endogenous expression), or after cell stimulation with MSU (stimulated expression). The concentration of cytokines was assessed by ELISA. In preeclamptic pregnant women, gene expression of NLRP1, NLRP3, caspase-1, IL-1β and TNF-α by monocytes stimulated or not with MSU was significantly higher than in NT and NP groups. Stimulation of monocytes from preeclamptic and non-pregnant women with MSU induced increased gene expression of NLRP3, caspase-1 and TNF-α in relation to the endogenous expression in these groups, while this was not observed in the NT group. The cytokine determination showed that monocytes from women with PE produced higher endogenous levels of IL-1β, IL-18 and TNF-α compared to the other groups, while the stimulus with MSU led to higher production of these cytokines in preeclamptic group than in the NT group. In conclusion, the results showed increased basal gene expression of NLRP1 and NLRP3 receptors in monocytes from PE group. These cells stimulation with MSU demonstrates that uric acid plays a role in NLRP3 inflammasome activation, suggesting the participation of this inflammatory complex in the pathogenesis of preeclampsia.

Preeclampsia is a pregnancy disorder characterized by imbalance between pro- and anti-inflammatory cytokines associated with high plasma levels of uric acid and Interleukin-1 beta (IL-1β). The inflammasome is a protein complex that mediates innate immune responses via caspase-1 activation promoting secretion of IL-1β and IL-18 in their active forms, and also release of the high-mobility group box 1 protein (HMGB1). As the placenta seems to play an important role in the pathogenesis of PE, the present study investigated the expression of genes and proteins related to the inflammasome in placentas from pregnant women with severe preeclampsia. Placental tissue was collected from 20 normotensive pregnant women and 20 preeclamptic women, and inflammasome components, NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3), caspase-1, IL-1β and IL-18, as well as tumor necrosis factor-alpha (TNF-α) and HMGB1 were evaluated by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and also quantified by reverse transcription-qPCR (RT-qPCR). Compared with normotensive pregnant women, placenta from women with PE showed a significant increase in NLRP3, caspase-1, IL-1β, TNF-α and HMGB1 mRNA. Immunohistochemical staining of NLRP3, caspase-1, IL-1β and TNF-α in placental villi, as well as the levels of caspase-1, IL-1β, TNF-α and HMGB1 in placental homogenate were significantly higher in the preeclamptic group than in the normotensive group. However, mRNA expression of IL-18 and its protein concentrations were lower in placentas from preeclamptic women. The results suggest that placentas from pregnant women with preeclampsia show higher expression of NLRP3 inflammasome, which may be involved in the exaggerated inflammatory state in preeclampsia.

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.

Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content.

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240 h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18 h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8 h PI. Hypoferremia peak was observed at 12 h and returned to normal levels at 30 h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6 h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1 µg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2 h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.