B. Serrano-Pérez

Maternal and fetal immune response patterns in heifers experimentally infected with Neospora caninum in the second trimester of pregnancy – A descriptive study

Fetal and maternal immune responses 3, 6 and 9 weeks post infection (wpi) were investigated in cows experimentally infected with Neospora caninum on day 110 of gestation. Descriptive analysis showed that the fetuses had lower percentages of spleen T cell subpopulations (CD3+, CD4+ and CD8+) at 6 wpi compared to 3 wpi and/or 9 wpi, with the lowest percentages observed in a dead fetus found upon euthanasia at that time. Increased expression of most cytokines over levels recorded at 3 and 9 wpi were found in fetuses that were alive at 6 wpi. Up-regulated Th1, Th2 and Treg expression was also observed at 6 wpi in the spleen and in the lymph nodes draining the placenta of the cows. At the placental level, while most cytokines were down-regulated from 6 wpi, up-regulation of IL-4 expression was observed at 6 wpi in the caruncle. Our results suggest that the immune response at 6 wpi was crucial for fetal survival in this model of bovine neosporosis.

Cytokine gene expression in aborting and non-aborting dams and in their foetuses after experimental infection with Neospora caninum at 110 days of gestation

Neospora caninum is a major cause of abortion in cattle. However, it is not known why not all infected animals abort. In this study, Th1 (IFN-γ), Th2 (IL4) and T reg (IL-10) cytokine gene expression was examined by real time PCR using the TaqMan approach in all of these dams and their foetuses after experimental infection with the isolate Nc-Spain7 at 110 days of pregnancy and euthanasia 6 weeks after infection. In prior published work, foetal death was observed in three of six infected dams and transplacental infection in all the 6 infected foetuses. In the spleen of the dams, IL-4 expression was down-regulated in dams with aborted/non viable foetuses compared to both uninfected dams (controls, n=3) and infected dams with live fetuses at euthanasia. In the lymph nodes draining the placenta, up-regulated expression of IL-4 was observed in infected dams with live foetuses compared to control dams. In the placenta, infected dams with live foetuses had significantly up-regulated IFN-γ in both caruncle and cotyledon and up-regulated IL-10 in cotyledon compared to control dams. Infected live foetuses showed up-regulated expression of IFN-γ and IL-10 in foetal spleen, and showed downregulated expression of IL-4 in the thymus compared to control uninfected foetuses. Expression of any cytokine in the thymus was significantly lower compared to the levels observed in foetal spleen. The results indicate an up-regulated expression of Th1, Th2 and Treg in infected dams with live foetuses and in their foetuses. On the other hand, down-regulation of Th2 immune responses and Treg cytokines were observed in infected dams which had aborted or had non-viable foetuses at euthanasia, suggesting an immunological recovery of cytokine gene expression levels in dams a few weeks after an abortion occurred.

Maternal and foetal cytokine production in dams naturally and experimentally infected with Neospora caninum on gestation day 110.

In the present study, IFN-γ (Th1), IL-17A (Th17) and IL-4 (Th2) concentrations in response to concanavalin (ConA) and Neospora caninum antigen (Nc-1) stimulation were determined in cultures of cells from control uninfected (n=4), naturally N. caninum-infected (n=3) and experimentally N. caninum-infected (n=6) pregnant dams and their foetuses. Experimental animals were infected at 110days of gestation and euthanized 6weeks post-infection. In culture supernatants from the dams, significantly higher IFN-γ and IL-4 levels were found in the experimentally-infected animals compared to the control or naturally-infected dams. However, among the experimentally-infected dams no significant differences in IFN-γ production were observed regardless of the incidence of live or aborted/dead foetuses, though spleen cultures of dams carrying live foetuses showed the highest levels of IFN-γ. IL-17A production was very low and occasional in the dams infected with N. caninum and did not seem to be a major regulator of IFN-γ production in this model. Experimentally infected dams with live foetuses showed higher IL-4 levels and accordingly IFN-γ/IL-4 ratios were significantly lower than ratios recorded for cows with aborted/dead foetuses. In the infected foetuses of these dams, only spleen cultures showed high levels of IFN-γ and IL-4 after Nc-1 antigen and ConA stimulation, respectively. No IL-17A was detected in the foetuses. As conclusion, although we could not clearly relate a protective immune response against N. caninum abortion only to IFN-γ levels in cell cultures, our results highlight the important role of an inverse IFN-γ/IL-4 balance in conferring protection against abortion induced by this parasite.

Experimental Neospora caninum infection modifies trophoblast cell populations and plasma pregnancy-associated glycoprotein 1 and 2 dynamics in pregnant dairy heifers.

Neospora caninum is an obligate intracellular protozoan that causes abortion in cattle worldwide. Plasma concentrations of pregnancy-associated glycoprotein 1 (PAG-1), produced by binucleate trophoblast cells, are used for pregnancy diagnosis and as a marker of foetal-placental well-being, while PAG-2, produced by both mono- and binucleate trophoblast cells, may serve as an indicator of abortion risk. In prior work, natural N. caninum infection was found to modify plasma PAG-1 and PAG-2 patterns. The present study examines PAG-1 and PAG-2 dynamics and trophoblast cell populations following experimental infection with N. caninum. The study population was comprised of 17 N. caninum seronegative Holstein-Friesian heifers. On day 110 of gestation, 6 heifers were inoculated intravenously with 10(7) taquyzoites of N. canimum. Plasma samples for PAG-1 and PAG-2 determinations were collected on days 0, 7, 14, 21 and 42 post infection. During the study course, pregnancy was normally expressed in all controls while three infected heifers lost their foetuses. All heifers were euthanised on day 42 post infection and placentome samples from the 14 non-aborting heifers were collected to examine trophoblast cell populations. Plasma PAG-1 and PAG-2 concentrations in non-infected heifers increased until the day of euthanasia while non-aborting infected heifers showed a temporary fall in PAG-1 (P<0.004) and PAG-2 (P<0.002) concentrations from 7 to 14 days post infection (dpi). The two dams aborting at 14 and 21 dpi and a third dam with a mummified foetus upon euthanasia showed dramatic PAG-1 and PAG-2 reductions from 14 dpi to undetectable levels upon euthanasia. A stereological study of placentomes revealed significantly higher relative proportions of mono- (P=0.035) and binucleate (P=0.029) trophoblast cells at 42 dpi in non-infected heifers than infected non-aborting heifers. According to our findings, following experimental N. caninum infection on day 110 of gestation, non-aborting heifers showed a brief reversible drop in plasma PAG-1 and 2 concentrations two weeks later and reduced proportions of bi- and mono-nucleate trophoblast cells 42 days after infection. In aborting dams, dramatically reduced PAG levels were related to severe placental damage and a non-viable pregnancy.

Crosstalk between uterine serpin (SERPINA14) and pregnancy-associated glycoproteins at the fetal-maternal interface in pregnant dairy heifers experimentally infected with Neospora caninum

Infection with Neospora caninum is the leading cause of abortion in cattle. In cows naturally infected with N caninum, plasma concentrations of pregnancy-associated glycoproteins (PAG) 1 and 2 indicate fetal-placental well-being, whereas an excess of progesterone in the second trimester of gestation has been related to high abortion rate. The immunosuppressive action of progesterone on the uterus during gestation has been attributed in part to the uterine serpins (SERPINA14). This study examines expression patterns of the genes SERPINA14, PAG, and PAG2 at the fetal-maternal interface in dairy heifers experimentally infected with N caninum during the second trimester of pregnancy, when most abortions takes place in natural conditions. Irrespective of infection, expression of SERPINA14 was higher, and expression of PAG1 and PAG2 lower, for intercaruncular endometrium than for caruncles or cotyledons. Cotyledonary tissues showed the highest expression of both PAG genes but lowest expression of SERPINA14. The expression of SERPINA14 was significantly higher in intercaruncular endometrium of control dams than for infected animals, pointing to potential disruption of modulation of maternal immune function during infection. Dramatically reduced SERPINA14 was particularly apparent in infected dams with aborted fetuses. There was also a negative association between N caninum antibody titers with SERPINA14 and PAG expression in infected animals, further suggesting that N caninum infection downregulates the uterine immunosuppressive function of SERPINA14.

Coxiella burnetii total immunoglobulin G, phase I and phase II immunoglobulin G antibodies, and bacterial shedding in young dams in persistently infected dairy herds

The current study examines Coxiella burnetii infection patterns in young dairy dams around the calving period in persistently infected high-producing dairy herds. Infection patterns were determined in terms of total immunoglobulin G (IgG) and phase-specific IgG antibodies by enzyme-linked immunosorbent assay and bacterial shedding by real-time polymerase chain reaction (qPCR). On days 171–177 of gestation, at parturition, and on days 15–21 and 91–97 postpartum, 7 first-parity cows and 7 second-parity cows were sampled for serology and qPCR. Total phase-specific I (PhI) and II (PhII) IgG antibodies were detected in 2 animals at days 171–177 of gestation. Four additional animals underwent seroconversion on days 91–97 postpartum. Three of 6 seropositive dams according to total IgG, showed a PhI+/PhII+ profile, whereas dams that seroconverted exhibited a PhI–/PhII+ (2/6) or PhI+/PhII– (1/6) profile. An indirect fluorescent antibody test for PhI and PhII immunoglobulin M (IgM) was performed on plasma samples from the shedding dams, confirming seropositivity in a first-parity dam that seroconverted, and detecting a sudden spike of PhI-IgM antibodies in 1 further dam. No relationship was detected in young C. burnetii–infected animals between total IgG, PhI and/or PhII antibodies, and bacterial shedding throughout the study period. The highest bacterial load measured by qPCR was recorded in a second-parity dam. This animal presented abnormal peripheral blood counts, which would be an indication of severe peripheral blood alterations in some infected cattle. This study suggests that young shedder cows are mostly seronegative in early stages of infection.

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy-Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG-1 are used for pregnancy diagnosis and as a marker of placental/foetal well-being, while those of PAG-2 may be an indicator of abortion risk in Neospora caninum-infected cows. Studies have shown that N. caninum infection modifies PAG-1 and PAG-2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG-1, PAG-2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG-2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non-infected foetuses. In infected foetuses, significantly higher concentrations of PAG-1 (p < 0.001) and PAG-2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non-infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG-1 and PAG-2 levels and pH values than fluids from non-infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.

The presence of two ovulatory follicles at timed artificial insemination influences the ovulatory response to GnRH in high-producing dairy cows

This study sought to examine the impact of the presence of two co-dominant (ovulatory) follicles at the time of artificial insemination (AI) on the ovulatory response to GnRH given in a fixed-time AI protocol. The study population comprised 622 lactating dairy cows: 306 (49.2%) with a single follicle, 198 (31.8%) with two bilateral follicles (one follicle per ovary) and 118 (19%) with two unilateral follicles (same ovary). Based on odds ratios, cows with two bilateral or unilateral follicles were less likely (by factors of 0.09 and 0.11, respectively) to undergo ovulation failure compared with cows with one follicle (P = 0.01 and P = 0.02, respectively); the likelihood of ovulation failure decreased 0.75 times with every one-mm increase in follicle diameter for cows with a single follicle, whereas individual follicle diameter was not related to ovulation failure in cows with two bilateral follicles (P = 0.001). The likelihood of double ovulation decreased 0.7 times with every one-mm diameter difference between the larger and smaller follicle for all cows with two follicles (P = 0.001), whereas cows with two unilateral follicles showed a higher (P < 0.05) double ovulation rate than cows with two bilateral follicles. In 116 (58.6%) of the cows with two bilateral follicles, only the larger follicle ovulated in 59.5% cows, whereas only the smaller one ovulated in the remaining 40.5% cows. In these cows, a one-mm size difference between the larger and the smaller follicle gave rise to a 1.12-fold increase in the ovulation failure rate for the larger follicles (P = 0.0001). Cows with two bilateral follicles were more likely (by a factor of 1.5) to conceive than cows with one follicle (P = 0.001). Significant right-left differences were not found in cows with two bilateral follicles, whereas the right ovary was more active than the left in the remaining cows. Our results indicate that cows with two co-dominant follicles at AI show different ovulation patterns to those with one dominant follicle. A higher rate of ovulation failure was observed among cows with one follicle than cows with two follicles, whereas the conception rate was higher for cows with two bilateral follicles than for the remaining cows. In cows with two follicles, double ovulations along with ovulation of the smaller follicle were related to the least size difference between the larger and smaller follicle.

Effect of PRID-Delta devices associated with shortened estrus synchronization protocols on estrous response and fertility in dairy cows

Abstract This study evaluated the effect of a once-used progesterone (P4) intravaginal device (PRID) associated with four different shortened P4-based estrus synchronization (ES) protocols on estrous response (ER) and pregnancy per AI (P/AI) in cyclic and acyclic lactating dairy cows. Cows (n=465) were randomly assigned to one of the following protocols: 1) 2PGG, cows were given a PRID-Delta and 100 μg GnRH i.m. at PRID insertion (day 0). The PRID was left for 5 d, and 25 mg of dinoprost (PGF2α) i.m. given twice at PRID removal and 24 h later; 2) 2PGGe, same treatments as 2PGG plus 500 IU of eCG i.m. at PRID removal; 3) 2PGe, same treatments as 2PGGe, except GnRH was not given at PRID insertion; 4) PGe, same treatments as 2PGe, except PGF was only given at PRID removal. A total of 258 cows received a new PRID-Delta containing 1.55 g of P4, whereas 207 cows received a once-used PRID. Estrus was determined from P4 device removal until 96 h after using an automated heat detection system. Cows in estrus were given a second GnRH at AI and those without signs of estrus by 96 h after PRID removal were given GnRH and timed-AI (TA I). All inseminations were performed by one technician with commercially available frozen-thawed semen. Ultrasonography was performed at initiation of protocol and 28-34 days post AI to determine cyclicity and pregnancy status, respectively. Cows receiving once-used P4 devices had greater ER than cows receiving a new device (59.9 vs. 50.0; P=0.029), but P/AI did not differ between P4 devices, respectively (P>0.1; 40.6 vs 40.7%). Cyclic cows were less likely to display estrus than acyclic cows by a factor of 0.66 (P=0.036). Cows subjected to the 2PGe (2.41; P<0.01) protocol were more likely to display estrus than cows subjected to the 2PGG, whereas cows subjected to the PGe protocol did not differ (0.94; P=0.8) from those in the 2PGG group. Despite differences in ER, neither cyclicity nor estrus synchronization protocol affected P/AI (overall 40.6%). In summary, cyclic cows, those given a new P4 device and those subjected to either 2PGG or PGe protocol had reduced ER. However, all the factors examined had no significant effect on P/AI. All the estrus synchronization protocols resulted in acceptable fertility.