Molecular genotyping and serological evaluation of Toxoplasma gondii in mothers and their spontaneous aborted fetuses in Southwest of Iran

BackgroundGiven the lack of routine screening and the high prevalence of toxoplasmosis in pregnant women in Iran, the current study aimed to find out the rate and features of Toxoplasma gondii infection in the spontaneously aborted human fetuses in Kohgiluyeh and Boyer-Ahmad Province, Southwestern Iran.MethodsThis cross-sectional study was performed on 100 spontaneously aborted fetuses’ tissues and their mother blood samples. The mothers’ sera were evaluated for anti-Toxoplasma antibodies while their buffy coat and aborted fetuses tissues were evaluated for Toxoplasma DNA. PCR product at GRA6 locus was sequenced and phylogenetic analysis was done. Likewise, quantitative Real-Time PCR was performed to find out the parasite burdens in mothers buffy coat and fetuses tissues.ResultsUsing serological method, anti-Toxoplasma IgG and IgM antibodies were detected in 7 (7%) and 3 (3%) out of 100 sera from women with spontaneous abortion. Real-time PCR method detected T. gondii DNA in the buffy coat of one seronegative and 2 (out of 3) IgM seropositive cases. None of the samples from aborted fetuses were infected with T. gondii. BLAST and phylogenetic analysis showed that the sequenced isolates belonged to type I of T. gondii and two identified T. gondii isolates were taxonomically grouped into one clade.ConclusionOur findings revealed type I genotype of T. gondii in two mothers with spontaneous abortion, without fetus involvement. It is necessary to examine more aborted fetuses’ samples from different geographical areas to determine the association between Toxoplasma genotype and abortion.     

Interferon-gamma expression and infectivity of Toxoplasma infected tissues in experimentally infected sheep in comparison with pigs

Livestock animals are a potential risk for transmission of toxoplasmosis to humans. Sheep and pigs still remain an important source because their meat is often eaten undercooked which has been regarded as a major route of infection in many countries. Moreover, porcine tissues are processed in many food products.In the current study, the IFN-gamma (T-helper 1 cells), IL-4 (Th2 cells) and IL-10 mRNA (Treg cells) expression by blood mononuclear cells, and the serum antibody response against Toxoplasma gondii total lysate antigen, recombinant T. gondii GRA1, rGRA7, rMIC3 and rEC2, a chimeric antigen composed of MIC2, MIC3 and SAG1, was studied in sheep the first two months after a T. gondii infection and compared with these responses in pigs. At the end of this period, the parasite distribution in heart, brain and two skeletal muscles in sheep was compared with this in pigs.Whereas the parasite distribution was similar in sheep and pigs, the antibody response differed considerably. In sheep, antibodies appeared against all tested T. gondii antigens, but mainly against rGRA7, rMIC3₂₃₄₃₀₇ and TLA whereas in pigs only rGRA7-specific antibodies could be demonstrated. Also, the cytokine response differed. Both in sheep and pigs an IFN-gamma response occurred which seemed to be a slightly more pronounced in sheep. In sheep, also IL-10 and IL-4 mRNA expression showed an increase, but later than IFN-gamma and with more variation. However, in pigs no such increase was seen.As concerning diagnosis, results indicate that serum antibodies against GRA7 in live sheep and pigs and heart tissue for bioassay and qPCR in slaughtered animals are the best targets to demonstrate presence of T. gondii infection.     

旋毛虫53kuES重组蛋白单克隆抗体的制备及其免疫金标试纸条的研制

为建立一种简易、快速检测动物血清中的旋毛虫排泄分泌(Excretory-secretory,ES)抗原的胶体金免疫层析试纸法,本研究选用旋毛虫53 ku ES重组蛋白免疫BALB/c小鼠,应用杂交瘤细胞技术制备单克隆抗体(MAb).利用辛酸-饱和硫酸铵法和亲和层析柱联合法纯化MAb及兔抗旋毛虫53 ku ES多克隆抗体,利用枸橼酸三钠还原法制备的胶体金颗粒标记MAb,制备免疫金标试纸条.结果表明,获得的2株IgG1型杂交瘤细胞系4D10和5A2能够稳定分泌抗体,且效价高;胶体金标记MAb 4D10所制备的试纸条具有重复性好、灵敏性和特异性高的优点;该试纸条4℃条件下可密封保存6个月以上.     

Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant

The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (±S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 ± 97.5) were 77.25% (724 ± 98) in group 1, 88.02% (381 ± 97.5) in group 2, 38.92% (1943 ± 130.3) in group 3, 44.31% (1771.4 ± 102) in group 5, 59.28% (1295.2 ± 99.1) in group 7 and 55.69% (1409.5 ± 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.     

Preparation and Primary Application of Immunity Colloidal Gold Test Strip for Fasciola gigantica

To establish a rapid, simple, sensitive and adapting field application assay for water buffalo Fasciola gigantica, the monoclonal antibodies of hybridoma cell strains 7D1 and 7D4 against excretory-secretary antigen(ES Ag) of Fasciola gigantica were prepared and purified. Meanwhile, the colloidal gold particle was prepared by the sodium cirrate reduction method, then the affinity purified 7D1 monoclone antibodies was labeled with the 30 nm colloidal gold particles. The 7D4 monoclonal antibodies and the goat anti-mouse IgG antibody using as secondary antibodies were coated on the surface of nitrocellulose filter(NC) membrane as the test line (T line) and control line (C line), respectively. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The test results showed the prepared strip had a detecting prescribed minimum of 0.94 μg/mL of Fasciola gigantica excretory-secretary antigen, and it had no any cross reaction with the antigens of Eurytrema pancreaticum, Paramphistome, Chlamydia, Toxoplasma gondii and Trypanosoma evansi. Using the prepared strip to investigate the 334 fresh feces from buffaloes in Guangxi and the positive rate was 38.62%. These results indicated that this strip method was simple, rapid and easy determination, good specificity, high sensitivity, and adapted field application assay for Fasciola gigantica.     

The role of rodents and other wildlife in the epidemiology of swine toxoplasmosis

Two swine production units and their contiguous wildlife populations were used for this study. These herds were located 7 miles apart in the major swine producing area of south central Georgia. Herd A had a Toxoplasma gondii antibody prevalence of 27.6% in all ages of swine sampled, with an increased incidence of 13.6% over a 5-month period. This herd was maintained under a semi-range condition. The swine in Herd B were maintained exclusively in concrete floored, enclosed buildings. This herd had a 0.85% T. gondii prevalence. Rodents and a few other wildlife and domestic species were trapped in or around both herds. These animals were examined for Toxoplasma antibodies using the same test procedure utilized for swine sera, the indirect immunofluorescent (IIF) test. Additionally, rodent tissues were homogenized and suspensions prepared for interperitoneal (I/P) inoculation into CF₁ laboratory mice.Rodents and wildlife species examined were: Mus musculus, Peromyscus leucopus, Rattus norvegicus, Sigmodon hispidus, Procyon lotor, and Didelphis marsupalis. Feral and domestic animals other than swine that also were tested for the presence of T. gondii antibodies included two cats, two horses, and a dog. The overall prevalence of T. gondii antibodies in all non-porcine species examined was 67% for those animals in and around the premises of Herd A, and 63% for those in and around Herd B.Toxoplasma infectivity of rodents whose tissues were processed and inoculated into laboratory mice correlated well with the results of the IIF test on the serum of these wild rodents. No tachyzoites of T. gondii were found in the peritoneal exudate of laboratory mice post I/P inoculation with wild rodent tissue, with one exception.While there was no significant difference in Toxoplasma infectivity in non-porcine species on these two premises, management practices appeared to be the determining factor in swine infection with T. gondii. Excluding wildlife precluded infection.     

大片形吸虫免疫胶体金检测试纸条的研制及初步应用

为建立一种快速、简便、适应现场应用的水牛大片形吸虫检测方法,本试验利用抗大片形吸虫ES抗原单克隆抗体杂交瘤细胞株7D1和7D4分别制备和纯化得到单克隆抗体7D1和7D4;采用柠檬酸三钠还原法制备颗粒大小为30 nm的胶体金,再用胶体金标记纯化单克隆抗体7D1,在硝酸纤维素膜的质控带和检测带处分别包被羊抗鼠IgG二抗和单克隆抗体7D4,经反应条件优化,组装成了大片形吸虫免疫检测试纸条。经测试该检测试纸条对ES抗原的检测下限为0.94 μg/mL,与胰扩盘吸虫、前后盘吸虫、支原体、弓形虫、伊氏锥虫均无交叉反应,特异性好。采集广西地区334份水牛新鲜粪样并用试纸条检测,阳性率为38.62%。结果表明,试纸条方法操作简单、快速、易于判定,特异性好,敏感性高,适合基层大面积普查和现场检测大片形吸虫。     

Distribution of cysts and tachyczoites in calves and pregnant cows inoculated with toxoplasma gondii oocysts

Sixteen calves and 6 cows were each inoculated with 100 000 infective oocytes of the GT-1 strain of Toxoplasma gondii. Cattle were necropsied between 3 and 287 days post-inoculation (DPI) and their tissues were inoculated into mice or fed to Toxoplasma-free cats for the detection of Toxoplasma in bovine tissues. Ten to 10 000-fold more T. gondii were recovered from small intestine and mesenteric lymph nodes of calves at 3 and 6 DPI than from lungs and liver, and the number of T. gondii in bovine tissues was reduced 1000-fold between 6 and 8 DPI. By using the pepsin digestion technique, or feeding tissues to Toxoplasma-free cats, it was demonstrated that T. gondii encysted in bovine tissues as early as 11 DPI and persisted as late as 287 DPI. More Toxoplasma gondii cysts occurred in livers than in any other bovine tissue. Of the 6 cows inoculated at 95–155 days after breeding, 5 delivered normal calves and T. gondii was isolated from only one of these calves. One cow was barren. Toxoplasma gondii was not isolated either by mouse inoculation or by feeding cats tissues from 2 cows killed 132 and 190 DPI. Toxoplasma gondii was not isolated in mice inoculated with tissues of cows killed 98 and 109 DPI, but cats fed on bovine tissues shed T. gondii oocysts. The organism, however, was isolated in mice inoculated with the mesenteric lymph nodes of 1 of the 2 cows killed 162 and 168 DPI, and from the small intestine of the other. Cats fed tissues of these cows later shed T. gondii oocysts.     

猫瘟热病毒胶体金快速检测试纸的研究

利用双抗体夹心和胶体金免疫层析技术的原理,在玻璃纤维膜、硝酸纤维膜的检测线(T)和对照线(C)上分别喷上胶体金与猫瘟热病毒(FPV)单克隆抗体(3C4)的偶联物、FPV多克隆抗体和羊抗鼠IgG单克隆抗体,制成检测FPV抗原的猫瘟热病毒胶体金快速检测试纸,对临床26份虎粪便样本进行检测,并与PCR检测法及韩国(ASAN ...     

The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18

More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P < 0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.     

莱克多巴胺胶体金免疫层析法快速检测试纸条的研制

为研制一种快速、简便的基于胶体金免疫层析法的试纸条,快速检测猪尿中莱克多巴胺（ractopamine,RAC药物残留）,采用柠檬酸钠还原法制备胶体金颗粒,标记莱克多巴胺单克隆抗体并喷于玻璃纤维上制备胶金垫,RAC-BSA偶联抗原和羊抗鼠IgG分别结合于硝酸纤维膜上,依次将样品垫、胶体金垫、硝酸纤维素膜和吸水纸组装并切割成莱克多巴胺胶体金免疫层析快速检测试纸条。试验结果表明该快速检测试纸条的检测限为5 ng/mL,可在8~10 min内完成测试,肉眼即可判断,无需其他检测设备。该试纸条灵敏度、特异性、稳定性和重复性均达到临床使用要求,适用于现场快速检测和筛选工作。     

A dot-immunobinding assay for infectious bronchitis virus

Common Whatman filter paper grade 1 and nitrocellulose membrane were compared for their sensitivity in a dot-immunobinding assay for detection of serum antibody titers to Arkansas avian infectious bronchitis virus (AIBV). For a blue to purple color detection, serum antibodies were bound to AIBV antigen adsorbed on the filter-paper discs or nitrocellulose membrane. Rabbit anti-chicken IgG horseradish-peroxidase (HRP) conjugate and hydrogen peroxide with 4-chloro-1-naphthol (HRP-color development reagent) were applied. The study indicates that very small amounts of antigen/antisera are needed for the dot-immunobinding assay. The test is sensitive, economical, and easy to run and can be completed within 6-8 hours.     

水牛伊氏锥虫病免疫胶体金试纸条的研制及初步应用

为建立一种快速、简便、适应现场应用的水牛伊氏锥虫病诊断方法,采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗伊氏锥虫VSG抗原的OB6单克隆抗体,在硝酸纤维素膜上包被纯化的抗伊氏锥虫VSG抗原的TB7单克隆抗体和兔抗小鼠IgG,作为检测带和质控带,然后优化条件,研制成检测伊氏锥虫病的免疫胶体金试纸条.通过交叉试验表明该试纸条与衣原体、弓形虫、旋毛虫、巴贝斯焦虫和大片吸虫无交叉反应.该试纸条最低可以检出1∶3 200倍稀释的伊氏锥虫VSG抗原(浓度为3.11 mg·mL-1).应用该试纸条对230份水牛血清样本进行初步检测,同时用ELISA做平行试验,阳性符合率为88.2％.结果证明该试纸条是一种快速、灵敏、特异的水牛伊氏锥虫病的检测方法,为水牛伊氏锥虫病的现场检测诊断和预控提供了有效的方法.     

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.     

Prevalence of anti-<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibodies in sport horses from Qazvin,Iran

In the present study, the prevalence of antibodies to Toxoplasma gondii in sport horses of Qazvin was examined using modified agglutination test (MAT). On 52 horse sera totally examined for anti-Toxoplasma antibodies, 37 horses (71.2%) were seropositive by MAT. Results of the present study showed a high rate of Toxoplasma infection in horses in Qazvin area. More comprehensive study on equine toxoplasmosis is recommended.     

Prevalence of Toxoplasma gondii infection in Belgian house cats

Five hundred and sixty seven sera of healthy house cats aged 3 months to 7 years, were examined for the presence of anti-toxoplasma antibodies by indirect immunofluorescence assay and compared to SAG1 and TLA enzyme linked immunosorbent assays as alternative test. Twenty-five percent of cats tested positive for IgG and/or IgM. Seroprevalence increased with age from 2% below 12 months of age up to 44% at age 7. Sensitivities of SAG1 and TLA ELISA were 84.1% and 88.6%, respectively. Peak levels in seroprevalence were correlated to increased IgG titers in TLA ELISA. Our results suggest that T. gondii infections are common in house cats and that there is a high chance for a negative cat to seroconvert in its second life-year.     

Bovine IgM and IgM response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen-antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.     

Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.     

Monoclonal antibodies to equine IgM improve the sensitivity of West Nile virus-specific IgM detection in horses

West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNV-specific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the three anti-IgM antibodies. The anti-IgM 1-22 mAb correctly identified all NVSL samples. Both the polyclonal antibody and monoclonal anti-IgM 2B-63 identified eight out of ten samples correctly. The three assays were then compared using serum samples from clinically healthy animals (n=33) and horses with neurological signs (n=21). High Spearman rank correlations (0.76-0.86) were found among the ELISA results. Inter-test agreements (weighted kappa) for assay interpretation resulted in strong agreement (0.95) of the results obtained by the mAbs and moderate agreements when monoclonal and polyclonal anti-IgM-based assays were compared. To determine the analytical sensitivities of anti-WNV IgM detection, serial dilutions of WNV IgM-positive serum samples were analyzed. The highest sensitivity was obtained by using the anti-IgM 1-22 mAb to capture IgM from equine serum. In conclusion, the use of monoclonal anti-IgM antibodies can improve the sensitivity of IgM detection in the acute phase of WN disease.