Association Between Plasma Homocysteine Levels and Subclinical Hypothyroidism in Adult Subjects: A Meta-Analysis.

Increased plasma homocysteine (Hcy) levels have been widely documented in patients with overt hypothyroidism; however, the significance of Hcy level changes in patients with subclinical hypothyroidism (SCH) remains controversial. The aim of this meta-analysis was to determine the Hcy status in patients with SCH compared with euthyroid subjects. We searched PubMed, Embase, and Cochrane Library databases prior to December 2019 to identify eligible studies and assessed the quality of selected studies using the Newcastle-Ottawa Quality Assessment Scale. Publication bias was evaluated by Begg's test and Egger's test. Meta-regression analysis was conducted to investigate the source of heterogeneity. A likely source of heterogeneity was the year of the study. All statistical analyses were performed with RevMan 5.3 and Stata 12.0 software. Our meta-analysis of twelve observational studies with 684 patients showed that those with SCH aged between 18 and 65 years old were associated with a slightly increased plasma Hcy level compared with euthyroid controls. The pooled result of the weighted mean difference (WMD) of increased tHcy levels was 1.16 µmol/l (95% CI: 0.51, 1.82; p=0.0005). The Hcy level in patients with SCH aged between 18 and 65 years old is significantly increased compared to euthyroid controls.

Evaluation of Neospora caninum truncated dense granule protein 2 for serodiagnosis by enzyme-linked immunosorbent assay in dogs.

Neosporosis is an infectious disease caused by Neospora caninum, and it primarily affects cattle and dogs. An infection by N. caninum causes fetal abortion and neonatal mortality. Previous proteomics and immunoscreening analyses revealed that N. caninum dense granule antigen 2 (NcGRA2) has potential for serodiagnosis of N. caninum. Consequently, we expressed the truncated NcGRA2 (NcGRA2t), which lacks a signal peptide. We compared the serodiagnostic performances of recombinant NcGRA2t with that of truncated surface antigen 1 of N. caninum (NcSAG1t). Specificity testing using sera from mice infected with Toxoplasma gondii indicated that the NcGRA2t recombinant protein does not cross-react with T. gondii. In addition, we detected anti-NcGRA2t antibody at the acute stage in experimentally infected dogs, while detecting anti-NcSAG1t antibody during both the acute and chronic stages. Our results suggest that the levels of anti-NcGRA2 antibody reflect parasite activation in dogs. In conclusion, antibodies against NcGRA2t and NcSAG1t are suitable indicators to distinguish the acute and chronic stages of N. caninum infection.

Interactive host cells related to Mycoplasma suis α-enolase by yeast two-hybrid analysis.

Mycoplasma suis belongs to the haemotrophic mycoplasmas, which colonise the red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. In addition to MSG1 protein, α-enolase is the second adhesion protein of M. suis, and may be involved in the adhesion of M. suis to porcine red blood cells (RBC). To simulate the environment of the RBC, we established the cDNA library of swine peripheral blood mononuclear cells (PBMC). The yeast two-hybrid (Y2H) system was adopted to screen α-enolase interactive proteins in the PBMC line. Alignment with the NCBI database revealed four interactive proteins: beta-actin, 60S ribosomal protein L11, clusterin precursor and endonuclease/reverse transcriptase. However, the M. suis α-enolase interactive proteins in the PBMC cDNA library obtained in the current study provide valuable information about the host cell interactions of the M. suis α-enolase protein.

Isolation, Identification, and Pathogenicity of Neospora caninum China Yanbian strain.

BACKGROUND: The aim of the study was to provide a point of reference to study the Neospora caninum infections in China. METHODS: Genome DNA was extracted from the brains of aborted fetuses and specific PCR was performed with N. caninum Nc5-targeted specific primers. Fetal bovine brain tissues were homogenized and continuously cultured in Vero cells with double antibodies. The medium was replaced at 2-d intervals and the state of cells was observed. RESULTS: A 608 bp Nc5 gene band was detected by PCR amplification. After sequencing, the sequence of the sample shared 99.5% homology with GenBank (AF061249). Brain homogenates were continuously cultured in Vero cells for 34 d and N. caninum was found. The results of IFAT and Nc5 gene-based PCR detection were N. caninum-positive, and the parasite was tentatively named N. caninum China Yanbian strain. BABL/c mice were inoculated with the separated parasites and showed clinical symptoms of ataxia and limb paralysis after 12 d. Only 3 mice survived. The blood of dying mice and the hearts, livers, spleens, lungs, kidneys, and brains of dead mice were collected aseptically. The Nc5 gene-based PCR showed that N. caninum may exist in brains, livers, and spleen. Based on immunohistochemical observations, we showed that N. caninum tachyzoites existed in the brains and livers. CONCLUSION: We have successfully isolated bovine-specific N. caninum strain from brain tissues of aborted cattle in the China Yanbian region. This isolated strain has a strong infectious ability towards BABL/c mice.

Generation and immunity testing of a recombinant adenovirus expressing NcSRS2-NcGRA7 fusion protein of bovine Neospora caninum.

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-γ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

Molecular detection of Theileria species in sheep from northern China.

Ovine theileriosis is a tick-borne disease that restricts the development of small ruminant husbandry in northern China. In this study, we report on a molecular epidemiological survey of ovine Theileria spp. in 198 blood samples taken from sheep in northern China. The DNA samples were screened by a nested polymerase chain reaction (PCR) targeting the 18S rRNA gene of ovine Theileria spp. The prevalence of ovine Theileria spp. in Yanji, Nongan, Longjing, Toudao and Jinchang was 80%, 40%, 37%, 24% and 32%, respectively. The sequencing analyses approved the present of the T. orientalis and/or T. luwenshuni in these regions. Taken together, we have demonstrated a high incidence of Theileria spp. in northern China that calls for the need to design effective control programs for ovine theileriosis.

Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model.

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.

[Screening, cloning and sequence analysis of the differential expression genes in Longissimus dorsi of Yanbian yellow cattle].

Annealing control primer (ACP) system was applied to find candidate genes related to lipidosis in muscle of Yanbian yellow cattle by screening differentially expressed genes (DEGs) in Longissimus dorsi, which had significant difference on intramuscular fat (IMF) content. Thirty steers, aged at 28 month-bullocks were selected to measure the IMF content in L. dorsi. Two groups of bullocks (three heads per group) with the highest and the lowest contents of IMF were selected to build a RNA pool, and DEGs of two groups were analyzed by ACP system. Twelve DEGs were identified and sequenced by amplification with 20 arbitrary primers (fragment sizes were 200-890 bp). In these genes, eight were already known as functional groups of cytoskeleton, cytokine signal transduction, protein synthesis, energy metabolism, and others, four were unknown. All the 12 ESTs were screened by ACP system, which may participated in regulating on lipidosis in muscle. This study established a foundation for further screening of lipidosis related genes.

Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice.

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.

Adjuvant effect of bovine heat shock protein 70 on piroplasm surface protein, p33, of Theileria sergenti.

In this study, we investigated the immunological effects of bovine heat shock protein 70 (HSP70) on the major Theileria sergenti surface protein (p33). The gene encoding p33 was expressed as a fusion protein with bovine HSP70 from a plasmid vector. The adjuvant function of HSP70 on p33 was evaluated with regard to antibody response, cytokine production, and a challenge experiment in mice or cattle. HSP-p33 fusion protein provoked higher humoral and cellular immunity than either Escherichia coli-expressed p33 or piroplasm soluble protein. The HSP adjuvant activity toward p33 was also possible to detect in the inoculated cattle. The overall growth of parasites in cattle was significantly restrained in the HSP-p33-inoculated group, up to 50-52 days longer than in the controls. The present results indicate that HSP-p33 fusion protein is a promising candidate vaccine for clinical theileriosis in the field.

Establishment of an efficient enzyme-linked immunosorbent assay for the detection of Eperythrozoon sius antibody in swine.

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.

Studies on the Small Body Size Mouse Developed by Mutagen N-Ethyl-N-nitrosourea.

Mutant mouse which show dwarfism has been developed by N-ethyl-N-nitrosourea (ENU) mutagenesis using BALB/c mice. The mutant mouse was inherited as autosomal recessive trait and named Small Body Size (SBS) mouse. The phenotype of SBS mouse was not apparent at birth, but it was possible to distinguish mutant phenotype from normal mice 1 week after birth. In this study, we examined body weight changes and bone mineral density (BMD), and we also carried out genetic linkage analysis to map the causative gene(s) of SBS mouse. Body weight changes were observed from birth to 14 weeks of age in both affected (n = 30) and normal mice (n = 24). BMD was examined in each five SBS and normal mice between 3 and 6 weeks of age, respectively. For the linkage analysis, we produced backcross progeny [(SBS × C57BL/6J) F1 × SBS] N2 mice (n = 142), and seventy-four microsatellite markers were used for primary linkage analysis. Body weight of affected mice was consistently lower than that of the normal mice, and was 43.7% less than that of normal mice at 3 weeks of age (P < 0.001). As compared with normal mice at 3 and 6 weeks of age, BMD of the SBS mice was significantly low. The results showed 15.5% and 14.1% lower in total body BMD, 15.3% and 8.7% lower in forearm BMD, and 29.7% and 20.1% lower in femur BMD, respectively. The causative gene was mapped on chromosome 10. The map order and the distance between markers were D10Mit248 - 2.1 cM - D10Mit51 - 4.2 cM - sbs - 0.7 cM - D10Mit283 - 1.4 cM - D10Mit106 - 11.2 cM - D10Mit170.

Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses.

An immunochromatographic test for the simultaneous detection of Babesia caballi- and B. equi-specific antibodies (BceICT) was developed using a recombinant B. caballi 48-kDa rhoptry protein (rBc48) and a recombinant truncated B. equi merozoite antigen 2 (rEMA-2t). An evaluation of the ability of the BceICT to detect antibodies in sera from uninfected horses and experimentally infected horses showed high sensitivities and specificities of 83.3% (10/12 sera) and 92.9% (52/56 sera), respectively, for the anti-B. caballi antibody and 94.1% (16/17 sera) and 88.2% (45/51 sera), respectively, for the anti-B. equi antibody. Results from the detection of antibodies in field-collected sera indicated that the BceICT results corresponded with those of enzyme-linked immunosorbent assays (ELISA), showing 91.8% correspondence (67/73 sera) for B. caballi and 95.9% correspondence (70/73 sera) for B. equi, and that the BceICT results also corresponded with the ICT for B. caballi and for B. equi, both of which were 98.2% (55/56 sera). The comparable results of the ICT and ELISA and the simplicity and rapidity of the performance of the ICT suggest that the BceICT would be a feasible test for the simultaneous serodiagnosis of both agents of equine babesiosis in the field.

Development of rapid immunochromatographic test with recombinant NcSAG1 for detection of antibodies to Neospora caninum in cattle.

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.

[Sequencing analysis of the control region and intron1 region and characterization of two novel microsatellites in obese gene of pig].

For the first time, a 16 kb fragment of the porcine Ob gene, namely intron1, exon1, 5' region of Ob gene, was restrictively analyzed and sequenced by the primers designed in the portion of the swine Ob sequence. The first small 38 bp untranslated exon1 is located 11.1 kb upstream of the initiator ATG codon, and two novel microsatellites SW200 and SW160 are found in intron1. Promoter analysis of several putative binding sites revealed that this 300 bp promoter located at -1 to -300, including C/EBP and two Sp1, may be as effective as the longer promoter in directing leptin transcription. To examine microsatellites association with important economic traits, we statistically analyzed genotypes and alleles of the two microsatellites. Statistical analysis carried out by SAS 8.2 revealed significant positive correlation between the two microsatellites genotypes and the litter size in first parity of Erhualian.

Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses.

An immunochromatographic test (BeICT) for the rapid detection of antibodies against Babesia equi was developed. It clearly differentiated B. equi-infected horses from B. caballi-infected and uninfected horses. The agreement with enzyme-linked immunosorbent assay results was 96.7% in the detection of field sera. The results suggest that BeICT is rapid, simple, reliable, and suitable for use to detect B. equi infection in the field.

Serodiagnosis of Babesia gibsoni infection in dogs by an improved enzyme-linked immunosorbent assay with recombinant truncated P50.

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.

Seroepidemiologic studies on Babesia equi and Babesia caballi infections in horses in Jilin province of China.

The prevalence of equine piroplasmosis caused by Babesia equi and Babesia caballi in northeast China has remained unknown, although the People's Republic of China is recognized as an endemic country for the diseases. In the present study, we investigated the prevalence of equine piroplasmosis in Jilin province, a part of northeast China. A total of 111 serum samples were taken from horses in eastern Jilin, and examined for diagnosis of B. equi and B. caballi infections by the enzyme-linked immunosorbent assays with recombinant antigens, equi merozoite antigen-1 and P48, respectively. Of the 111 samples, 38 (34%) and 36 (32%) samples were sero-positive for B. equi infection and B. caballi infection, respectively. In addition, 14 (12%) samples were sero-positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in northeast China.