Pathogenesis of bovine neosporosis

The protozoan parasite Neospora caninum is a major pathogen of cattle and dogs, being a significant cause of abortion in cattle in many countries. It is one of the most efficiently transmitted parasites, with up to 90% of cattle infected in some herds. The pathogenesis of abortion due to Neospora is complex and only partially understood. Losses occur after a primary infection during pregnancy but more commonly as the result of recrudescence of a persistent infection during pregnancy. Parasitaemia is followed by invasion of the placenta and fetus. It is suggested that abortion occurs when primary parasite-induced placental damage jeopardises fetal survival directly or causes release of maternal prostaglandins that in turn cause luteolysis and abortion. Fetal damage may also occur due to primary tissue damage caused by the multiplication of N. caninum in the fetus or due to insufficient oxygen/nutrition, secondary to placental damage. In addition, maternal immune expulsion of the fetus may occur associated with maternal placental inflammation and the release of maternal pro-inflammatory cytokines in the placenta. Thus N. caninum is a primary pathogen capable of causing abortion either through maternal placental inflammation, maternal and fetal placental necrosis, fetal damage, or a combination of all three. The question of how N. caninum kills the fetus exposes the complex and finely balanced biological processes that have evolved to permit bovine and other mammalian pregnancies to occur. Defining these immunological mechanisms will shed light on potential methods of control of bovine neosporosis and enrich our understanding of the continuity of mammalian and protozoal survival.     

Vaccination of mice with chitosan nanogel-associated recombinant NcPDI against challenge infection with Neospora caninum tachyzoites

The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n. route, 80% of the mice were protected. Association of recNcPDI with the alginate-coated nanogels protected all mice against disease. Quantification of the cerebral parasite burden showed a significant reduction of parasite numbers in most experimental groups vaccinated i.n., except those vaccinated with alginate-mannose nanogels with or without recNcPDI. For i.p. vaccinated groups, no significant differences in cerebral infection densities were measured, but there was a reduction in the groups vaccinated with recNcPDI associated with both types of nanogels. Analysis of the immune responses of infected mice indicated that association of recNcPDI with nanogels altered the patterns of cytokine mRNA expression profiles, but had no major impact on the antibody subtype responses. Nevertheless, this did not necessarily relate to the protection.     

Variables Associated with Infections of Cattle by Brucella abortus., Leptospira spp. and Neospora spp. in Amazon Region in Brazil

The frequency of Neospora spp., Leptospira spp. and Brucella abortus infections in adult cattle was determined in herds of the State of Pará, Brazil, which is an important region for cattle production located in the Amazon region. A total of 3466 adult female cattle from 176 herds were tested, leading to a frequency of seropositive animals of 14.7%, 3.7% and 65.5% and a herd positivity of 87.4%, 41.3% and 98.8% for infections caused by Neospora spp., B. abortus and Leptospira spp., respectively. The five most frequently diagnosed serologic responses to Leptospira spp. were those against serovars hardjo, wolfii, grippotyphosa, hebdomadis and shermani. The following associations were found: practice of artificial insemination, large farm size, large herd size, large number of dogs and high number of total abortions per year with the presence of antibodies against serovar hardjo; positive results to serovar grippotyphosa with the presence of dogs; inappropriate disposal of aborted foetuses with positivity to serovar hebdomadis. Serovar grippotyphosa was also associated with number of episodes of abortions. Neospora spp. positive herds were associated with episodes of abortion and B. abortus infection with the disposal of dead animals and aborted foetuses on pastures and with the use of artificial insemination. In conclusion, the high frequency of brucellosis, leptospirosis and neosporosis in the region may be a consequence of social, natural and raising conditions as: (i) climate conditions that favour the survival and spread of pathogens in the environment; (ii) farms located in regions bordering forest areas; (iii) farms in areas of difficult access to the veterinary service; (iv) extensive beef herds raised at pastures with different age and productive groups inter‐mingled; and (v) minimal concerns regarding hygiene practices and disease prevention measures.     

新孢子虫速殖子在HCT-8、Vero和Hela细胞中培养的比较

目的观察和比较新孢子虫速殖子在人结肠癌细胞(HCT-8)、人宫颈癌细胞(Hela)和非洲绿猿肾细胞(Vero)中的生长情况。方法分别用HCT-8、Hela和Vero细胞(RPMI-1640培养基)连续培养新孢子虫速殖子9d,观察并计数速殖子数量,绘制生长曲线;4d取培养物作吖啶噔染色,用激光共聚焦显微镜观察速殖子。结果新孢子虫速殖子用HCT-8、Vero和Hela细胞培养均生长,尤以在HCT-8和Vero细胞中生长较快,第6d虫体数达到高峰,且在HCT-8中高峰期可持续2d,在Vero中生长高峰可持续1d。在Hela细胞中速殖子生长较慢,虫体数量一直处于较低水平。吖啶噔染色观察,HCT-8和Vero细胞中纳虫泡较大且速殖子数量较多,Hela细胞内纳虫泡较小且速殖子数量较少。结论新孢子虫速殖子在HCT-8细胞中生长较快,纳虫泡较大且速殖子数量较多。HCT-8细胞可以替代Vero细胞用于速殖子的体外培养。     

The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5′ coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3′ UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5′ coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.     

The role of the colostrum and milk in <Emphasis Type="Italic">Neospora caninum</Emphasis> transmission

Summary Neospora caninum, an apicomplexan protozoan causes economical losses due to reproductive failure associated with abortion among cattle. The transmission of N. caninum is possible through vertical transmission in utero, or according to the modern nomenclature endogenous and exogenous infection modes and horizontal transmission through ingestion of oocysts. Limited data is available on the vertical transmission during suckling time, via colostrum and milk. In this paper the main scientific aim focused on N. caninum DNA detection in the milk and colostrum of seropositive cows have been reviewed. In this term, the risk of animals and humans infection has been discussed.     

The in-vitro interaction between several components of the canine immune system and infective larvae of Ancylostoma caninum

In this paper, in-vitro experiments are reported which indicate that in dogs infected with the hookworm Ancylostoma caninum , there exists a cooperation between specific IgG antibodies and fixed complement components (C3). This relation is important in the adherence of peripheral blood leucocytes to the surface of the larvae. Fixation of complement occurs via the alternative pathway.     

Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory I secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES1–6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30kDa; AC/ES 2 recognized a broad band ranging from 50–200kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.     

新孢子虫NcSAG4基因克隆及原核表达

目的构建新孢子虫NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中表达。方法根据新孢子虫NcSAG4基因序列设计特异性引物,以新孢子虫总核酸为模板PCR扩增目的片段,与pMD-18-T连接,构建克隆载体pMD-NcSAG4,经双酶切后回收目的片段,与表达载体pGEX-T连接,构建原核表达载体pGEX-NcSAG4,用IPTG诱导,通过SDS-PAGE及Western blot进行鉴定。结果成功构建了新孢子虫NcSAG4基因原核表达载体pGEX-Nc-SAG4;SDS-PAGE电泳显示,在IPTG诱导下阳性菌高效表达了分子质量单位为18.79ku的蛋白质;Western blot显示表达产物可被抗新孢子虫的多克隆血清识别。结论成功构建了NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达,为建立经济、实用的诊断新孢子虫病的ELISA方法奠定了基础。     

Analysis of the immune response to Neospora caninum in a model of intragastric infection in mice

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll-like Receptor 4- and functional IL-12Rbeta2 chain-deficient C57BL/10 ScCr mice were challenged intragastrically with 5 x 10(6) N. caninum tachyzoites. All parasite-inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 x 10(7) N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B- and T-cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN-gamma and IL-10 mRNA were detected in both groups of infected BALB/c mice compared with non-infected controls, whereas in the Peyer's patches only IFN-gamma mRNA levels were found to be increased. Parasite-specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum-specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.