Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P<0.05 or P<0.01) from those of vaccinated ewes that neither contracted brucellosis nor excreted the pathogen. In general, all the tests detect the infection in the non-vaccinated ewes with substantial effectiveness. It can be concluded that the high levels of circulating antibodies and of antigen-specific gamma-IFN are related to active Brucella infection. Similarly, the state of protection against the disease, but not necessarily against infection, due to a previous immunization with the Rev. 1 vaccination, appears to be responsible for a low level of detectable immune response. Nevertheless, the design of the study limits conclusions to pregnant ewes and cannot be extrapolated to non-pregnant ewes or rams. Likewise, the study provides no information on animals which are carriers of B. melitensis.     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other -proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Immune response in rams experimentally infected with Brucella ovis

Cellular as well as humoral immune responses were detected in six rams experimentally infected with Brucella ovis. Specific antibodies were detectable by enzyme-linked immunosorbent assay by day 11 after infection in all the rams. The levels of IgM antibodies and total antibodies in the serum rose until 33 and 41 days after infection respectively, then levelled off. Antigen-induced blastogenic responses by lymphocytes developed as early as five days after infection in all rams but had decreased to low levels by day 63 in most. Blastogenesis induced by phytohaemagglutinin and concanavalin A varied among infected rams and did not differ significantly (P greater than 0.05) from control rams. All rams had developed delayed-type skin hypersensitivity by day 63 after infection. One ram which did not become infected as a result of exposure had low levels of B ovis serum antibodies and a detectable antigen-induced lymphocyte blastogenic response before infection, suggesting the involvement of cell-mediated immunity in protection against B ovis.     

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30 dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240 dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation.     

Teladorsagia circumcincta antibodies in serum and milk samples in experimentally infected lactating ewes

The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r=0.3; p<0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r=-0.2; p<0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r=0.2; p<0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions.     

羊布氏杆菌的分离与鉴定

从一患睾丸炎公山羊中分离到一株革兰氏阴性短小杆菌,该菌经柯兹洛夫斯基鉴别染色红色,在普通绵羊血琼脂、巧克力平板上生长缓慢,对该菌的16S rDNA进行PCR扩增,测序,BLAST比较,结果显示该菌与羊布氏杆菌的16S rDNA有100%的同源性,菌体与布氏杆菌标准阳性血清起强凝集反应。结果显示,该分离菌为布氏杆菌。     

Effect of feeding lasalocid to pregnant ewes experimentally infected with Toxoplasma gondii.

Sixteen 50 day gestational ewes were fed lasalocid at the rate of 30 g t-1 and were orally inoculated with 100 infective Toxoplasma gondii oocysts 5 days after beginning feeding of lasalocid. Seventeen control ewes were similarly inoculated with T. gondii and were not fed lasalocid. The rate of abortion and neonatal mortality in both treated and untreated ewes was similar, indicating that feeding lasalocid was not effective in preventing T. gondii abortion in sheep.     

羊种布鲁氏菌分子生物学研究进展

综述了近年来羊种布鲁氏菌的研究概况,着重介绍了羊种布鲁氏菌基因组、致病因子、致病机理以及分子生物学检测方法等方面的研究进展,并对未来的研究发展趋势提出见解,为最终使该菌所引发的相关疾病得到有效控制提供参考.     

羊布鲁氏菌甲酰基转移酶的生物信息学研究

本研究旨在分析和预测羊布鲁氏菌甲酰基转移酶基因及其编码蛋白的结构与功能,用于指导其生物学功能的实验室研究。综合利用生物信息学软件(DNAMAN和Vector NTI)、数据库(prosite和PDB)和网络服务器对甲酰基转移酶进行基本性质分析、三维结构预测和功能预测,展示了甲酰基转移酶的氨基酸分布情况,二级结构中形成螺旋、折叠、卷曲的氨基酸区段及结构域;预测并综合阐释了B细胞表位的柔韧性、表面可及性和亲水性参数。生物信息学预测和分析结果可为研究在布鲁氏菌致病及机体免疫应答中甲酰基转移酶的结构与功能的关系提供丰富资料。     

Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.