Prevalence of shedding and antibody to Coxiella burnetii in post-partum dairy cows and its association with reproductive tract diseases and performance : a pilot study

The bacterium Coxiella burnetii has been associated with reproduction disorders in dairy cattle. A cross-sectional study was conducted in Québec, Canada, to estimate the prevalence of C. burnetii in dairy cows from C. burnetii RT-PCR-positive and/or ELISA-positive herds. As a secondary objective, the associations between C. burnetii-positivity and three reproductive outcomes (purulent vaginal discharge, cytological endometritis, and success at first service) were assessed. A total of 202 post-parturient dairy cows from nine herds were sampled at 35 ± 7 days in milk. Vaginal mucus and composite milk were collected from each cow and screened for the presence of C. burnetii by real-time PCR (RT-PCR) and ELISA, respectively. Purulent vaginal discharge and cytological endometritis were evaluated using a Metricheck device and a modified cytobrush, respectively. The first insemination postpartum was done following an ovulation synchronization protocol around 70 days in milk, and success at first service was recorded. Multilevel logistic regressions adjusted for parity were used to model purulent vaginal discharge, cytological endometritis and success at first service according to C. burnetii cow status. All 202 RT-PCR-assayed vaginal samples were C. burnetii-negative. A positive result for anti-C. burnetii antibodies detection in composite milk was obtained in 25/202 samples and a doubtful result in 4/202 samples. After adjustment for sampling weights, the 202 ELISA-assayed composite milk samples gave an estimated overall prevalence of C. burnetii positive cows of 12.9 % (CI = 6.1–19.6 %) and of doubtful cows of 1.4 % (CI = 0.0–3.3 %). The proportion of ELISA-positive cows was lower in first parity (0%) compared to second (17.1 %) or third parity cows (20.0 %). The associations between ELISA positivity and reproductive outcomes were not statistically significant, perhaps due to the limited sample size, but could be used as pilot estimate for large-scale studies investigating the impact of C. burnetii infection on reproduction disorders in dairy cattle

Epidemiological study of Coxiella burnetii in dairy cattle and small ruminants in Québec, Canada

The bacterium Coxiella burnetii (C. burnetii) can infect a wide range of animals, most notably ruminants where it causes mainly asymptomatic infections and, when clinical, it is associated with reproductive disorders such as abortion. It is also the etiological agent of Q fever in humans, a zoonosis of increasingly important public health concern. A cross-sectional study was performed to estimate the apparent prevalence and spatial distribution of C. burnetii positivity in dairy cattle and small ruminant herds of two regions of Québec, Canada, and identify potential risk factors associated with positivity at animal and herd levels. In dairy cattle herds, individual fecal samples and repeated bulk tank milk samples (BTM) were collected. In small ruminant herds, serum and feces were sampled in individual animals. ELISA analyses were performed on serum and BTM samples. Real-time quantitative PCR (qPCR) was done on fecal and BTM samples. An animal was considered C. burnetii-positive when at least one sample was revealed positive by ELISA and/or qPCR, while a herd was considered C. burnetii-positive when at least one animal inside that herd was revealed positive. None of the 155 cows had a qPCR-positive fecal sample, whereas 37.2 % (95 % CI = 25.3–49.1) of the 341 sheep and 49.2 % (95 % CI = 25.6–72.7) of the 75 goats were C. burnetii-positive. The apparent prevalence of C. burnetii-positive herds was 47.3 % (95 % CI = 35.6–59.3) in dairy cattle herds (n = 74), 69.6 % (95 % CI = 47.1–86.8) in sheep flocks (n = 23) and 66.7 % (95 % CI = 22.3–95.7) in goat herds (n = 6). No spatial cluster of positive herds was detected. At the individual level, the only significant association with positivity in multivariable regressions was higher parity number in small ruminants. At the herd level, the use of calving group pen, the distance to the closest positive bovine herd, and small ruminant herd density in a 5 km radius were associated with dairy cattle herd positivity, whereas small ruminant herds with more than 100 animals and with a dog on the farm had greater odds of C. burnetii positivity. Our study shows that the infection is frequent on dairy cattle and small ruminant herds from the two studied regions and that some farm and animal characteristics might influence the transmission dynamics of the C. burnetii infection

Bringing patient advisors to the bedside: a promising avenue for improving partnership between patients and their care team

This paper presents an innovative model of care, which brings patients who have already been through a similar experience of illness (patient advisors) directly to the bedside of patients, where they are viewed as full-fledged members of the clinical team. As part of a pilot project, three patient advisors were recruited and met with patients who had sustained a traumatic amputation and were admitted to the only center of expertise in replantation of the upper limb in Canada. Several individual interviews and focus groups with patients and patient advisors have revealed very promising results. Indeed, patients have expressed tremendous appreciation for their meetings and interactions with patient advisors. They have stated feeling less isolated, having a better morale and increased hopefulness regarding the outcome of the care pathway. Patient advisors also felt a positive impact of their involvement. A larger study needs to be conducted to determine the impact of this model of care on patient adherence to treatment and on members of the health care team

Prevalence of Coxiella burnetii seropositivity and shedding in farm, pet and feral cats and associated risk factors in farm cats in Quebec, Canada

Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms

The MEK/ERK Pathway Promotes NOTCH Signalling in Pancreatic Cancer Cells

<div><p>Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome. </p> </div

Activation of the MEK/ERK pathway promotes NIC1 association with its transcriptional partners.

<p>MIA PaCa-2 cells were left untreated (-) or treated with EGTA (4mM) for 15min (+). EGTA-containing medium was removed and replaced by normal culture media (Ca²⁺) containing DMSO, PMA (100nM) or PMA + U0126 (10μM) for 90min. A. NIC1, MAML1 and CSL expression levels were assessed by western blot using the appropriate antibodies. <b>B</b>. CSL and MAML1 were immunoprecipitated (IP) prior to western blot analyses using the indicated antibodies. <b>C</b>. CSL was immunoprecipitated (IP) followed by western blot analyses of NIC1 (left). A graphic representation depicting the relative amount of NIC1 co-immunoprecipitated with CSL (NIC1/CSL) is shown (right). <b>D</b>. MAML1 was immunoprecipitated (IP) followed by western blot analyses of NIC1 (left). A graphic representation depicting the relative amount of NIC1 co-immunoprecipitated with MAML1 (NIC1/MAML1) is shown (right). <b>C</b>. and D. The results are the means ± SEM of at least three independent experiments. * p<0.05 compared to DMSO-treated cells. </p

NIC1 is phosphorylated in pancreatic cancer cells.

<p>NIC1 was immunoprecipitated (IP NIC1) from total lysates of exponentially growing MIA PaCa-2 (MIA), MIA PaCa-2 cells treated with EGTA (4mM) for 15min and then returned to their normal culture media for 90min, or exponentially growing BxPC-3 (Bx). Following NIC1 immunoprecipitation, phosphatase assays were performed (+) using the lambda phosphatase (λ phosphatase). The IP NIC1 not incubated with the lambda phosphatase is indicated as pNIC1. A representative Western blot using an antibody against NIC1 is shown.</p

Activation of the MEK/ERK pathway promotes NOTCH signalling.

<p><b>A</b>. MIA PaCa-2 cells were pre-treated (+) or not (-) with DAPT (25μM) overnight. Cells were then treated with PMA (100nM) for the indicated time periods. <b>B</b>. MIA PaCa-2 cells were left untreated (-) or treated with EGTA (4mM) for 15min (+). EGTA-containing medium was removed and replaced by normal culture media (Ca²⁺) containing DMSO, PMA (100nM) or PMA + U0126 (10μM) for the indicated time periods. <b>A</b>. and B. Cells were lysed and Western blot analyses were performed using the indicated antibodies. <b>C</b>. From similar experiments presented in B., a graphic representation depicting relative HES1 expression, for each time period, in treated cells (PMA, PMA+U0126) compared to control cells (DMSO-treated) is shown. <b>D</b>. From similar experiments presented in B., a graphic representation depicting relative c-MYC expression, for each time period, in treated cells (PMA, PMA+U0126) compared to control cells (DMSO-treated) is shown. <b>C</b>. and D. The results are the means ± SEM of at least three independent experiments. * p<0.05, ** p<0.005, ***p<0.0005 compared to the corresponding time period in DMSO-treated cells. # p<0.05, ## p<0.005, ### p<0.0005 compared to the corresponding time period in PMA-treated cells. </p

Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells.

<p><b>A</b>. HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total ERK1/2 expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. <b>B</b>. HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. <b>C</b>. NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.</p