Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.     

Fungi isolated from bovine udders,and their possible sources

AIM: To identify fungi isolated from infections of the bovine mammary gland, and establish their possible sources.

METHODS: From a herd of 420 cows, milk samples were collected from all quarters at calving and cultured to detect causative organisms. Quarters identified as infected with fungi were further sampled during early lactation. Samples from feedstuffs, the feed pad and ends of teats were also collected and analysed for the presence of fungi.

RESULTS: Eleven of 420 cows were diagnosed with intramammary infections (IMI) caused by yeasts (nine cows, 10 quarters) and moulds (two cows, three quarters). Six of the yeast species had previously been reported as being responsible for mastitis. Elevated somatic cell counts (SCC) were observed in many quarters, but most infections were eliminated spontaneously. Two of the fungi isolated from milk samples were also isolated from feedstuffs and teat swabs, and seven other fungi isolated from milk samples were not isolated from feed, the feed pad or cows' teats.

CONCLUSIONS: Isolation of fungi from the udder is rarely reported in dairy cows in New Zealand. In this herd, contamination of the end of the teat originating from feedstuffs and possibly exacerbated by the use of a feed pad may have led to the establishment of IMI caused by fungi.

CLINICAL RELEVENCE: Fungi are infrequently if ever reported in mastitis trial data or surveys in New Zealand and are probably of little clinical significance.     

Assessment of Different Functional Parameters of Frozen–Thawed Buffalo Spermatozoa by Using Cytofluorimetric Determinations

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen–thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR‐14/propidium iodide staining; mitochondrial function by JC‐1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC‐PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ^high), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %‐DFI, ‐DFI, SD‐DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome‐reacted live (ARL) and acrosome‐reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca²⁺ ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen‐thawed semen fertilizing potential.     

Simulation study of the competitive ability of erect, semi-erect and prostrate cowpea (Vigna unguiculata) genotypes

Ecophysiological simulation models provide a quantitative method to predict the effects of management practices, plant characteristics and environmental factors on crop and weed growth and competition. The INTERCOM interplant competition model was parameterised, calibrated by monoculture data for three cowpea (Vigna unguiculata) genotypes that differed in growth habit, common sunflower (Helianthus annuus) and common purslane (Portulaca oleracea), and used to simulate competition of cowpea cover crops with sunflower or purslane. The simulation results were compared with observations from field competition experiments in 2003 and 2004. INTERCOM more accurately simulated actual field data for the competition of cowpea genotypes and sunflower than companion field experiments for the competition of cowpea and purslane. The validated simulation model of cowpea and sunflower at two densities was used to study the effects of cowpea growth habit on final biomass production of cowpea and sunflower. The model suggested that erect growth habit was more competitive than semi‐erect and prostrate growth habit, when cowpea genotypes were grown with sunflower. Cowpea leaf area distribution was important to higher cowpea biomass production, while cowpea height growth was important to reduce sunflower biomass. Our simulation approach is suggested as a method for crop breeders to gauge the likely success of selection for competitive crops before undertaking expensive long‐term breeding experiments.     

Effect of short‐term exposure of cumulus–oocyte complex to 3‐morpholinosydnonimine on in vitro embryo development and gene expression in cattle

Short‐term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short‐term exposure of bovine mature cumulus–oocyte complex (COC) to 3‐morpholinosydnonimine (SIN‐1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN‐1 (0, 0.1, 1, 10 and 100 μM SIN‐1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN‐1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short‐term exposure of mature bovine COCs to SIN‐1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.     

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Gonadotropin‐induced Puberty Does Not Impair Reproductive Performance of Gilts over Three Parities

The aim of this study was to evaluate the reproductive performance of three parities of gilts treated or not treated with gonadotropin to induce puberty. Sixty gilts received 600 IU of equine chorionic gonadotropin (eCG) followed by 2.5 mg of porcine luteinizing hormone (LH) 72 h later. Fifty‐nine other gilts were exposed only to a mature boar for 15 min twice daily. Artificial insemination (AI) was performed at 0, 12 and 24 h after the detection of oestrus, and gestation was confirmed by ultrasound after 35 days. Sows were inseminated at the first post‐weaning oestrus. The total numbers of piglets born, piglets born alive, stillborn, mummified foetuses, as well as pregnancy and farrowing rates were evaluated for each of the three parities. Culling rates, farrowing intervals and weaning‐to‐oestrous intervals (WEI) were also analysed. Mean age at puberty and oestrous manifestation were not significantly different between treatments (p = 0.0639; 179.20 ± 17.52 compared with 173.96 ± 16.94, 91.66% compared with 94.92%) across the experimental period. However, females that underwent puberty induction showed modest increases both in the number of total pigs born and in the number of piglets born alive. In conclusion, puberty induction through exogenous gonadotropin administration in field conditions did not induce a more concentrated first oestrous manifestation, but trended to a modest increase in the number of pigs born alive in the first parity and a reduced culling rate during the first gestation.     

Genetic and phenotypic aspects of early reproductive performance in Raeini Cashmere goats

This study used pedigree information and data collected from 1979 to 2012 at the Raeini Cashmere goat breeding station, located in Baft City in Kerman Province in southeastern Iran. Genetic and phenotypic parameters for early reproductive traits of breeding does, including total numbers of kids born at first kidding (LSB1), total numbers of kids weaned at first kidding (LSW1), total birth weight of all kids born at first kidding (LWB1), total weaning weight of all kids weaned at first kidding (LWW1), and age at first kidding (AFK), were estimated using a Bayesian approach via Gibbs sampling. Posterior means for heritability estimates of LSB1, LSW1, LWB1, LWW1, and AFK were statistically significant, with values of 0.12, 0.23, 0.17, 0.15, and 0.46, respectively. Low-to-moderate additive genetic variation was present for the studied reproductive traits. Estimated genetic correlations among LSB1, LSW1, LWB1, and LWW1 were statistically significant and ranged from 0.12 between LWB1 and LWW1 to 0.72 between LSB1 and LSW1. Corresponding phenotypic correlation estimates were also statistically significant and ranged from 0.04 between LWB1 and LWW1 to 0.55 between LSB1 and LSW1. Posterior means of genetic and phenotypic correlations between AFK and other studied traits were statistically significant only for LSB1 and LWB1. For LSB1, LSW1, LWB1, and LWW1, we conclude that genetic and phenotypic improvement in any of these traits in Raeini Cashmere does would favorably influence all of the other traits. However, does that first kidded at younger ages have smaller litters at birth and lower litter birth weights at their first parity.

     

The histology of dermal glands of mating Breviceps with comments on their possible functional value in microhylids (Amphibia: Anura)

During mating male and female Breviceps become ‘glued’ together. The distribution of multicellular dermal glands varies between the sexes. The mate has a large number of holocrine glands on the ventrum, while the female has similar glands on the dorsum of the back. II thus appears that both sexes contribute to the ‘glueing’ mechanism. New hypotheses to explain the adhesion between the sexes are proposed, viz., that the shift to terrestrial habils and subterranean laying in Breviceps have secondarily resulted in male si2e diminution. In other microhylids which adhere during aquatic oviposition, yet demonstrate a terrestrial morphology and habits, bouyancy is suggested to be of primary functional value.