B. Serrano

Factors Affecting Plasma Pregnancy-associated Glycoprotein 1 Concentrations Throughout Gestation in High-producing Dairy Cows

This study was designed to establish the factors, if any, which could affect plasma pregnancy-associated glycoprotein-1 (PAG-1) expression in a study population of 87 pregnant, high-producing dairy cows. The factors examined were: semen providing breed (Holstein-Friesian vs Limousin), outcome of gestation (male vs female newborn, and singleton vs twin pregnancies), lactation number, milk production at pregnancy diagnosis, plasma progesterone concentration, season of gestation (warm period, March-November vs cool period, December-February), and day of gestation (40, 90, 120, 150, 180 and 210). Pregnancy was diagnosed by transrectal ultrasound on day 40 post-insemination and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The relative contributions of the different factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. No significant effects of the herd, foetal sex, milk production, lactation number and plasma progesterone concentrations were observed. In contrast, twin pregnancy, the use of Limousin semen and conception during the cool period were correlated with significantly increased plasma PAG-1 concentrations throughout gestation. Our data indicate that both cow well-being during early placental development, determined in our conditions by reduced heat stress when conception occurred in the cool season, and crossbreed pregnancies lead to improved PAG-1 production throughout the gestation period.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

BackgroundCoxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes.ResultsTo assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples.ConclusionsThis newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

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.

Dynamics of Coxiella burnetii antibodies and seroconversion in a dairy cow herd with endemic infection and excreting high numbers of the bacterium in the bulk tank milk

Possible factors related to seropositivity and seroconversion to Coxiella burnetii were examined in a dairy herd with a high Coxiella-seroprevalence and high excretion levels of the bacterium in the bulk tank milk. Antibodies were detected by using a commercial ELISA test in 50.7% of 603 parous cows. The likelihood of C. burnetii seropositivity significantly increased by factors of 1.87 and 1.61 for cows in their first and second terms of pregnancy, respectively, compared to non-pregnant cows. In 478 cows tested twice 12 months apart, the seroconversion rate was 5%. The likelihood of C. burnetii-seroconversion was 2.27 times lower in multiparous than in primiparous cows and 6.88 times higher in cows during their first 90 days in milk than dry-off cows. Our findings indicated a higher seroprevalence in the first and second terms of pregnancy, and that seroconversion mainly occurred in primiparous cows during their first 90 days in milk.

Coxiella burnetii Shedding During the Peripartum Period and Subsequent Fertility in Dairy Cattle

The objective of this study was to assess the effects of Coxiella burnetii shedding or seropositivity on post-partum recovery and subsequent fertility in high-producing dairy cows. Given the difficulty in diagnosing C.xa0burnetii infection at the farm level, an exhaustive series of tests in 43 pregnant animals that delivered at least one live calf were conducted, including blood serology and PCR of milk or colostrum, cotyledons (only at parturition), faeces, vaginal fluid against C.xa0burnetii on gestation Day 171-177, at parturition and on Days 1-7, 8-14, 15-21, 22-28, 29-35 and 90-97 post-partum. During scheduled herd visits, ultrasonography (US) of the genital tract and examination of vaginal fluid were performed on Days 15-21 (V1), 22-28 (V2), 29-35 (V3) and 51-57 (V4) post-partum by the same veterinarian. Logistic regression analysis revealed that the likelihood of suffering endometritis (the presence of echogenic intrauterine fluid (IUF), cervical diameter of ≥4xa0cm or endometrial thickness ≥0.75xa0cm) was lower in C.xa0burnetii-seropositive animals (ORxa0=xa00.10), compared with C.xa0burnetii-seronegative animals. According to Kaplan-Meier survival analysis, C.xa0burnetii-seronegative and non-shedding cows showed a delayed return to luteal activity and conception was delayed in non-shedding animals, compared with the remaining animals. Overall, the results of our study provide useful insight into the effects of C.xa0burnetii infection on post-partum recovery and subsequent fertility. In particular, animals not infected with Coxiella seem to be susceptible to infection and not protected against the bacterium in dairy herds. The elevated costs of determining an infection at the farm level, make monitoring of cows virtually impossible from a clinical point of view.

Anomalous Pregnancies during Late Embryonic/Early Foetal Period in High Producing Dairy Cows

This study analyses anomalous cases of gestation ending in pregnancy loss during the early foetal period and their effect on progesterone and plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations. Data derived from a large-scale ultrasound pregnancy diagnosis programme in high producing dairy cows. Over a 3-year period (2004-2007), a very low incidence (0.5%: 15 of 3094) of anomalous pregnancies was recorded. The results revealed that the following anomalies were detected on days 35-41 of gestation in cows carrying singletons with one single corpus luteum: embryo death in eight cows (0.3%); and embryo in the uterine horn contralateral to the corpus luteum in seven cows (0.2%). All these animals suffered pregnancy loss during the early foetal period. In cows carrying dead embryos, no signs of conceptus degeneration were observed on pregnancy diagnosis. Amnion size (approximately 25 mm diameter) and uterine horn fluid contents were estimated to be similar to those of the normal pregnant cows in this period. In the contralateral gestations, live embryos were observed in all ultrasound checks before pregnancy loss. Uterine fluid contents increased in the two cows in which gestation continued for more than a week. In the cases of embryo death but not in those of contralateral gestation, a drop in PAG-1 levels was noted prior to pregnancy loss. Two cows carrying dead embryos increased with time allantoic fluid contents. The PAG-1 values increased with time in one cow bearing a dead embryo (from 2.31 to 6.79 ng/ml) and in two of the contralateral gestations (from 1.66 to 2.33 ng/ml and from 0.39 to 6.79 ng/ml, respectively). Results of this study indicate that the foetal membranes continue to undergo some activity following embryo death, and that contralateral pregnancy may determine failure of the gestation process.

Serological and shedding patterns after Coxiella burnetii vaccination in the third gestation trimester in dairy cows.

This study sought to assess the effects of an inactivated phase I vaccine against Coxiella burnetii at the start of the third trimester of gestation on serological profiles, bacterial shedding patterns and subsequent reproductive performance in dairy cows. Cows were randomly assigned to a control (n = 78) or a vaccinated (n = 78) group on days 171-177 of gestation. Samples of placenta and colostrums at parturition, vaginal fluid, faeces, milk (PCR identification) and blood (anti-C. burnetii antibody detection) were obtained on the day of treatment and on days 91-97 post partum, and also on parturition day and weekly on days 1-7, 8-14, 15-21, 22-28 and 29-35 post partum in a subset of 70 animals. By Kaplan-Meier survival analysis, no significant effect of vaccination was detected on any of the reproductive variables studied. According to the odds ratio, C. burnetii shedding on days 171-177 of gestation was highly correlated with seropositivity against C. burnetii (OR = 9.1), while vaccination was not linked to reduced shedding of the bacterium. In shedders compared to others, the likelihood of pregnancy to first AI decreased and increased by factors of 0.26 and 16.1 on days 1-35 and 91-97 post partum, respectively. In conclusion, when administered at the start of the third trimester of pregnancy, the inactivated C. burnetii phase I vaccine failed to reduce bacterial shedding.

NO DETECTABLE PRECOLOSTRAL ANTIBODY RESPONSE IN CALVES BORN FROM COWS WITH COTYLEDONS POSITIVE FOR COXIELLA BURNETII BY QUANTITATIVE PCR

Samples from 45 dams (milk/colostrum, faeces, vaginal fluid and blood on days 171-177 of gestation and at parturition, and cotyledons at parturition) and their calves (blood collected before colostrum intake and weekly until days 29-35) were analysed to examine the vertical transmission of Coxiella burnetii and links between shedding and seropositivity. All calves were born C. burnetii seronegative. Only those born to seropositive dams seroconverted following colostrum intake. Logistic regression analyses indicated that the likelihood of dam seropositivity was 21 and 4.85 times higher for multiparous than for primiparous (65.6% vs. 8.3%, P = 0.006) and for prepartum shedding cows (75% vs. 38.2%, P = 0.03) compared to the remaining animals, respectively. In conclusion, the results of this study indicate no detectable precolostral antibody response in calves born from dams with cotyledons positive for C. burnetii by qPCR. In order to analyse the possibility of persistent infection due to immunotolerance to an early in utero infection, further studies will need to test for C. burnetii DNA. In addition, in the present study multiparous cows showed a significantly higher seroprevalence than primiparous cows and heifers, colostral antibodies were efficiently transferred to newborn calves, and there was a link between bacterial shedding on days 171-177 of gestation and Coxiella seropositivity of the dam.

Peripheral white blood cell counts throughout pregnancy in non-aborting Neospora caninum-seronegative and seropositive high-producing dairy cows in a Holstein Friesian herd

Pregnancy is characterized by transient changes in the maternal immune system, also evident at peripheral level. The present study analyzes the kinetics and possible factors affecting peripheral white blood cell populations throughout pregnancy in a herd of high-producing dairy cows chronically infected or not with Neospora caninum. We examined 54 pregnant parous cows: 29 Neospora-seronegative and 25 Neospora-seropositive cows. Blood samples were collected on Days 90, 120, 150, 180 and 210 of gestation. General Linear Model (GLM) repeated measures analysis of variance showed that the interaction Neospora-seropositivity × parity significantly affected total leukocyte, neutrophil and monocyte counts with lower levels of total leukocytes, lower neutrophil and higher monocyte counts recorded in primiparous Neospora-seropositive cows. In addition, N. caninum-seropositive cows had significantly increased monocyte counts on Day 180 of gestation compared to seronegative ones. Other factors significantly associated with changes in total and/or differential leukocyte profiles were period of pregnancy, season, twin pregnancy and milk production. In conclusion, a parity-associated effect of chronic N. caninum infection was observed on peripheral blood cell profiles in dairy cattle during gestation.