Seroprevalence of Toxoplasma gondii in mainland and sub-Antarctic New Zealand sea lion (Phocarctos hookeri) populations

AIMS: To investigate the seroprevalence of antibodies to Toxoplasma gondii in New Zealand sea lions (Phocarctos hookeri), as a potential contributor to reproductive failure.

METHODS: Archived sera were sourced from New Zealand sea lions from two recolonising mainland populations in the Otago Peninsula (n=15) and Stewart Island (n=12), as well as a declining population at Enderby Island (n=28) in the New Zealand sub-Antarctic. Sera were tested for antibodies to T. gondii using a commercially available ELISA (with samples considered positive if the sample to positive ratio was?>30%), and latex agglutination test (LAT; with titres ≥1:32 considered positive). Western blot analysis was used to validate the results of a subset of 14 samples.

RESULTS: Five samples from sea lions in mainland locations were confirmed positive for antibodies to T. gondii. Two adult females exhibited high LAT antibody titres (min 1:2048, max 1:4096) on both occasions when sampled 1 and 2 years apart, respectively. No animals from Enderby Island were seropositive.

CONCLUSIONS: Toxoplasma gondii infection is unlikely to be a major contributor to poor reproductive success in New Zealand sea lions. However, continued surveillance is pertinent to assess subclinical and clinical impacts of the parasite on these threatened populations. The commercial tests evaluated here, with further species-specific threshold refinement could provide a fast, inexpensive and reliable indicator of T. gondii exposure in New Zealand sea lions.     

Construction and Identification of Cell Penetrating Peptides VP22 Gene Fused with Enhanced Green Fluorescent Protein and Three Antigens of Toxoplasma gondii

To study a novel vaccine of Toxoplasma gondii,the recombinant plasmids that cell penetrating peptides VP22 gene fused with one enhanced green fluorescent protein（EGFP)(pcDNA-VP22-EGFP)and three antigens of T.gondii（pcDNA-VP22-SAG1,pcDNA-VP22-GRA4 and pcDNA-VP22-AMA1）were constructed,respectively.The recombinant plasmids were identified by PCR,restriction endonuclease digestion and DNA sequencing.The pcDNA-VP22-EGFP plasmid was transfected into COS7 cells,and the expression of VP22-EGFP in COS7 cells was confirmed by fluorescence microscope and RT-PCR.The results showed that all recombinant plasmid were constructed correctly.Green fluorescence was observed successfully under the fluorescence microscope in transfected cells after 72 h.The gene fragment of 1 449 bp was obtained by RT-PCR.The results indicated that the recombinant plasmids were constructed successfully.     

Detection of Zoonotic Protozoa Toxoplasma gondii and Sarcocystis suihominis in Wild Boars from Spain

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti‐Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP‐PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.     

弓形虫主要表面抗原P30基因克隆与表达

将液氮保存的弓形虫 Ｎ Ｔ 株经小鼠复壮后，取腹腔液提取弓形虫基因组 Ｄ Ｎ Ａ，采用 Ｐ Ｃ Ｒ 方法从弓形虫 Ｎ Ｔ 株中扩增出约 ８００ ｂｐ 的片段。产物经 Ｅｃｏ Ｒ Ｉ和 Ｘｂａ Ｉ酶切后，克隆到 ｐ Ｕ Ｃ１９ 载体中，构建了 ｐ Ｂ Ｖ Ｐ３０ 非融合表达质粒和 ｐ Ｅ Ｔ Ｐ３０ 融合表达质粒。ｐ Ｂ Ｖ Ｐ３０ 转化到宿主菌 Ｄ Ｈ５α、ｐ Ｅ Ｔ Ｐ３０ 转化到宿主菌 Ｂ Ｌ２１ （ Ｄ Ｅ３）后，分别经温控诱导和 Ｉ Ｐ Ｔ Ｇ 诱导，产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分析，ｐ Ｂ Ｖ Ｐ３０ 未发现表达产物，ｐ Ｅ Ｔ Ｐ３０ 出现约 ３０ ０００ 的产物。 Ｗ ｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 显示，该蛋白与兔抗弓形虫血清发生特异性反应；薄层扫描显示，该蛋白占菌体总蛋白的 ２０％ 以上。     

弓形虫MIC1蛋白在杆状病毒系统中的表达及活性分析

为构建可溶性表达弓形虫MIC1蛋白质的重组杆状病毒株并分析重组蛋白质的活性，通过PCR扩增弓形虫mic1基因的编码序列并连接到pFastBac 1质粒中，将重组的转移质粒转化DH10Bac感受态细胞，通过蓝白斑筛选获得重组杆粒，转染Sf9细胞后连续培养3代获得重组杆状病毒株。结果显示，Sf9细胞在感染后的第3天出现典型的细胞病变；间接免疫荧光和Western blot试验结果表明MIC1重组蛋白质在感染的Sf9细胞中成功可溶性表达；纯化的MIC1重组蛋白质不仅具有结合唾液酸乳糖的能力，同时能够刺激Balb/c小鼠产生较高水平的特异性抗体（>1∶25 600）。以上结果表明，通过杆状病毒表达系统可获得具有生物学活性的弓形虫MIC1重组蛋白质。     

Cross-sectional survey on Toxoplasma gondii infection in cattle, sheep and pigs in Serbia: seroprevalence and risk factors

Toxoplasmosis is a globally distributed zoonosis with a clinical impact in the unborn fetus and in the immunosuppressed individual. In Serbia, studies of risk factors for Toxoplasma gondii infection in humans have shown that the relatively high prevalence is associated mainly with consumption of undercooked meat and/or meat products. However, data on T. gondii infection in domestic animals mostly used for human consumption are scarce. We thus conducted a cross-sectional survey on the seroprevalence of T. gondii infection in a representative sample of cattle, sheep and pigs from different regions of Serbia between June 2002 and June 2003, and analyzed the main risk factors associated with the infection. Sera from 611 cattle (yearlings and adults of both sexes), 511 ewes, and 605 pigs (market-weight and sows), were examined for T. gondii antibodies by the modified agglutination test. The seroprevalences determined were 76.3% in cattle, 84.5% in sheep and 28.9% in pigs. The antibody levels ranged from 1:25 to 1:400 in cattle, and up to 1:25,600 in sheep and to 1:12,800 in pigs. Among the seropositive, the proportion of high antibody levels (> or =1:1600), suggestive of acute infection, was 10% in sheep, and 4% in pigs. Possible association of the infection with biologically plausible risk factors including gender, age, herd size/farm type, type of housing, feeding practices and region, was analyzed by univariate analysis, and variables significant at P< or =0.1 were included in multivariate logistic regression models. The results showed that risk factors for cattle were small herd size (odds ratio, OR=2.19, 95% confidence interval, CI=1.28-3.75, P=0.004) and farm location in Western Serbia (OR=2.04, 95% CI=1.10-3.79, P=0.024), while housing in stables with access to outside pens was protective (OR=0.37, 95% CI=0.21-0.67, P=0.001). In sheep, an increased risk of infection was found in ewes from state-owned flocks (OR=4.18, 95% CI=2.18-8.00, P<0.001) vs. private flocks, and, interestingly, also in those from Western Serbia (OR=4.66, 95% CI=1.18-18.32, P=0.028). In pigs, the risk of infection was highly increased in adult animals (OR=3.87, 95% CI=2.6-5.76, P<0.001), as well as in those from finishing type farms (OR=3.96, 95% CI=1.97-7.94, P<0.001). In addition to providing data on the current T. gondii seroprevalence in meat animals in Serbia, the results of this study show the main risk factors associated with infection, thereby pointing to the type of preventive measures to reduce T. gondii infection.     

弓形虫代谢分泌抗原疫苗对山羊流产高发区的免疫效果试验

制备以弓形虫E/SA（代谢分泌抗原）疫苗为主,辅以衣原体疫苗,对天祝县流产高发区的299只四群适龄母山羊（对照1群）在配种前进行免疫注射效果观测。结果在产羔期结束后统计流产率较往年显著降低,免疫后的三群羊分别由上年流产率的42%、36.7%、35%降为24.39%、0%和22.88%,流产率较上年度分别降低了41.92%、100%、34.62%,未免疫的对照群却上升了52.7%（37%上升为56.5%）。经现场考察和血清学分析,排除多种因素的干扰,证明弓形虫E/SA疫苗在本次免疫中效果显著。     

弓形虫重组GRA3抗原单克隆抗体的制备与鉴定

为了鉴定弓形虫特异性重组GRA3蛋白的抗原表位,以原核表达并纯化的融合蛋白GRA3免疫BALB/c小鼠,利用快速免疫法进行免疫,通过ELISA进行筛选,获得8株能够稳定分泌抗GRA3蛋白特异性单克隆抗体(McAb)的杂交瘤细胞。这8株McAbs亚类鉴定均为IgG型,特异性强,ELISA检测结果均为阳性,抗体效价均大于1∶200 000。该结果为进一步探索GRA3蛋白的结构和功能以及建立诊断方法奠定了基础。所生产的McAb可用于免疫组化和疫苗的制备,并可大大提高诊断的特异性。     

成都市宠物犬弓形体血清抗体调查

本研究对成都市宠物犬中弓形体的血清抗体进行了调查。结果表明,成都市宠物犬中抗体总体阳性率达18.1%,说明成都市宠物犬中弓形体的感染普遍,应引起足够的重视。