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.     

15种中草药对体外新孢子虫抑制效果的比较研究

[目的]筛选抗新孢子虫效果较好的中药。[方法]用新孢子虫虫体感染非洲绿猴肾细胞(Vero),观察细胞生长状态,并通过台酚蓝染色试验,计算各孔虫体死亡率和相对抑制率。[结果]黄芩、百部、天麻、黄连、槟榔、鸦胆子对体外培养的新孢子虫具有较好的杀灭和抑制作用。[结论]该研究为新孢子虫病的临床治疗提供依据。     

牛新孢子虫NcSAG1基因工程亚单位疫苗的免疫试验

为了解新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗对实验动物的免疫效果,本试验提取延边黄牛新孢子虫野毒株基因组DNA,用PCR技术扩增新孢子虫NcSAG1表面蛋白基因,并在大肠杆菌中进行原核表达,将Western-blot鉴定具有免疫活性的重组蛋白与弗氏佐剂混合制备NcSAG1基因工程亚单位疫苗,免疫BALB/c小鼠,应用间接ELISA方法测定体液免疫水平,应用流式细胞技术测定细胞免疫水平,以此评价疫苗对实验动物的免疫应答反应。结果显示,制备的NcSAG1基因工程亚单位疫苗免疫BALB/c小鼠后,在三免后第3天时,检测抗体的OD450nm值达0.688;CD4+/CD8+值达3.650,均显著高于重组蛋白免疫组和PBS对照组,说明制备的基因工程亚单位疫苗能够提高实验动物的体液免疫和细胞免疫水平。本试验为牛新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗的深入研究奠定了基础。     

Factors affecting plasma prolactin concentrations throughout gestation in high producing dairy cows

The aim of the present study was to investigate possible relationships between plasma concentrations of prolactin and the following factors throughout gestation in lactating dairy cows: photoperiod, season, milk production, Neospora caninum-seropositivity, twin pregnancy, and plasma concentrations of pregnancy associated glycoproteins-1 (PAG-1), progesterone and cortisol. Pregnancy was diagnosed by trans-rectal ultrasonography on Day 40 post-insemination and by palpation per rectum on Days 90, 120, 150, 180 and 210 or until abortion in aborting cows. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The study population was comprised of 73 non-aborting (54 Neospora-seropositive cows) and 20 aborting cows (all Neospora-seropositive) cows. In non-aborting cows, GLM repeated measures of analysis of variance revealed that lactation number and days in milk had no effect on plasma prolactin concentrations throughout gestation, whereas high plasma prolactin concentrations were significantly associated with high plasma levels of cortisol and PAG-1, with Neospora-seropositivity, positive photoperiod (increasing day length), twin pregnancy, and with low plasma progesterone concentrations. An interaction among plasma prolactin, plasma cortisol and milk production was observed in that plasma prolactin concentrations differed significantly throughout gestation and were highest in high-producing cows with high cortisol levels. In Neospora-seropositive non-aborting versus aborting cows, mean prolactin concentrations failed to differ on Days 40, 90, 120, 150 and 180 of pregnancy, whereas prolactin values were significantly lower (P = 0.03) in aborting animals on Day 210 of pregnancy. Our results indicate that a positive photoperiod and Neospora-infection lead to increased plasma prolactin concentrations throughout gestation. Reduced prolactin concentrations detected in Neospora-seropositive aborting cows compared to non-aborting animals suggests a protective effect of prolactin in N. caninum infection.     

犬新孢子虫NcSRS2基因原核表达质粒的构建

根据已发表的犬新孢子虫NcSRS2基因序列,设计1对含有EcoRⅠ和NotⅠ酶切位点的引物。以提取犬新孢子虫虫体基因组DNA为模板,应用PCR扩增获得NcSRS2 ORF基因片段,将此基因片段克隆到pMD18-T Simple载体上,用EcoRⅠ和NotⅠ双酶切该片段,回收得到含有两个酶切位点黏端的Nc-SRS2 ORF基因,将此基因片段克隆至相同酶切回收后的PGEM-4T-2原核表达载体中,获得重组质粒pGEX-NcSRS2,经PCR鉴定,限制性内切酶分析和克隆片段序列测定比较,证实了重组质粒的正确性。     

Factors associated with variation in Neospora caninum bulk-milk S/P ratios in initially bulk-milk negative testing Dutch dairy herds

We conducted a study on 81 initially bulk-milk ELISA negative dairy herds taken from a random sample of Dutch dairy herds to evaluate variation in bulk-milk S/P ratios and to study reasons for bulk-milk conversion. These herds were repeatedly (3-month intervals) tested between April 2004 and August 2005 and serostatus of all animals had previously been established as negative (N), low-positive (LP) or high-positive (HP). Of these herds, herd- and test-related factors associated with variation in sample over positive (S/P) ratios were analysed using a multivariable linear-mixed model with ‘herd’ as random effect. In addition, changes of animal serostatus in converting herds were described. S/P ratios were calculated as the optical density of the bulk-milk sample minus the optical density of the negative serum control divided by the difference in optical density between the positive and negative serum control.

Sixteen bulk-milk conversions in 12 dairy herds were seen with few indications of serological conversion in lactating cattle except for one herd in which recrudescence of infection appeared likely in nine cows. The effect of HP serostatus on bulk-milk S/P ratio was 2–3 times stronger compared with LP serostatus. In addition, bulk-milk S/P-ratio increased when the proportion of HP animals between 1 and 60 days in milk increased and decreased when the average milk-production level of the herd increased. Besides these herd-related factors, the use of different ELISA-testkits between test rounds had a significant effect on the S/P-ratio in bulk-milk samples.     

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

A prospective study of the effect of Neospora caninum and BVDV infections on bovine abortions in a dairy herd in Arequipa, Peru

We used a prospective seroepidemiological approach to investigate endemic abortion in a dairy herd in Arequipa, Peru, and its association with Neospora caninum and bovine viral-diarrhoea virus (BVDV) infections. Between January 2002 and March 2004, 1094 pregnancies were confirmed in 538 cows. Of these, 137 pregnancies (13%) in 121 cows ended in abortion. The serological status to N. caninum was assessed using a single serological screening, whereas BVDV status was assessed at the herd level through consecutive samplings of young stock.

Cox proportional-hazards models were used to estimate the effect of N. caninum and BVDV on the hazard of early (between day 42 and day 100 in gestation), and late (after day 100) abortions, respectively. Serological status to N. caninum was included as a dichotomous variable, and the effect of BVDV estimated at the herd level, as a time-dependent seasonal effect. Because data from repeated pregnancies were included, we considered possible lack of independence between observations and included frailty effects into the models. Our models also considered the possible confounding by parity and animal origin.

Only multiparity was associated with the hazard of early abortion (HR = 2.8 compared to nulliparous heifers). N. caninum seropositivity significantly affected the hazard of late abortion, but interacted with parity. The HRs for Neospora-positive animals were 6.4, 3.7 and 1.9, respectively, for nulliparous heifers, first-lactation cows and multiparous cows. Evidence of BVDV circulating (or not) among the young stock was not associated with abortions, but few cows in this herd were susceptible to incident infection.