Search

Toxoplasma gondii is an obligate intracellular protozoan parasite which seriously threatens the health of domestic animals and humans. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts greater than 200 nucleotides, which are widely involved in transcriptional and epigenetic regulations. However, little is known about the roles of host lncRNAs in the response to T. gondii infections. In this study, using Illumina sequencing technology, we analyzed the expression profiles of mRNAs and lncRNAs in BALB/c mouse brain following infection by T. gondii PRU strain (type II genotype) cysts. The identified differentially expressed (DE) RNAs were subjected to bioinformatics analysis. A total of 2,090 annotated lncRNAs along with 3,577 novel lncRNAs were identified. In the acutely infected mouse brain, a total of 330 mRNAs and 19 lncRNAs were dys-regulated, whereas 136 DE mRNAs and 9 DE lncRNAs were identified in chronically infected mouse brain. GO analysis revealed that these DE mRNAs identified at acute infection stage were involved in immune response, whereas DE mRNAs found at chronic infection stage were mostly enriched in response to protozoan. KEGG analysis showed that DE mRNAs were significantly enriched in disease related pathways. In addition, the putative mRNA-lncRNA co-expression network was constructed, and several hub regulatory RNAs were identified based on the transcriptome data. This study firstly characterized the co-expression profile of mRNAs and lncRNAs in mouse brain infected with T. gondii and provided a framework for further studies of the roles of lncRNAs in host neuropathology during toxoplasmosis progression.

Enterocytozoon bieneusi is a zoonotic parasite which is considered to be an opportunistic pathogen of humans and animals. A number of studies have reported E. bieneusi infection in various animals. However, no information is available on the occurrence of E. bieneusi in Tan sheep, a unique indigenous sheep species in the Ningxia Hui Autonomous Region, China. The objectives of the present study were to examine the prevalence and identify the genotypes of E. bieneusi in Tan sheep in China. A total of 1014 fecal specimens of Tan sheep from six farms in the Ningxia Hui Autonomous Region were examined by nested PCR amplification of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. The total prevalence of E. bieneusi was 12.2% (124/1014), ranging from 0.5 to 22.2% on six farms. Sequence analysis identified 10 genotypes of E. bieneusi, including three known genotypes, BEB6, COS-I, and CHG13, and seven novel genotypes designated as NX1 to NX7, which all belonged to group 2 by phylogenetic analysis. This is the first report describing the prevalence of E. bieneusi in Tan sheep, and the new genotypes identified in the current study expand the genotype distribution of E. bieneusi. These findings provide baseline data and have implications for the epidemiology and control of E. bieneusi infection in Tan sheep.

Background Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. Urine is an easily obtained clinical sample that has been widely applied for diagnostic purposes. However, changes in the urinary proteome during T. gondii infection have never been investigated. Methods Twenty four-hour urine samples were obtained from BALB/c mice with acute infection [11 days post infection (DPI)], mice with chronic infection (35 DPI) and healthy controls, and were analyzed using a label-free liquid chromatography tandem mass spectrometry analysis. Results We identified a total of 13,414 peptides on 1802 proteins, of which 169 and 47 proteins were significantly differentially expressed at acute and chronic infection phases, respectively. Clustering analysis revealed obvious differences in proteome profiles among all groups. Gene ontology analysis showed that a large number of differentially expressed proteins (DEPs) detected in acute infection were associated with biological binding activity and single-organism processes. KEGG pathway enrichment analysis showed that the majority of these DEPs were involved in disease-related and metabolic pathways. Conclusions Our findings revealed global reprogramming of the urine proteome following T.gondii infection, and data obtained in this study will enhance our understanding of the host responses to T. gondii infection and lead to the identification of new diagnostic biomarkers. Graphical Abstract

In this study, the genomes of three Plutella xylostella granulovirus (PlxyGV) isolates, PlxyGV-W and PlxyGV-Wn from near Wuhan and PlxyGV-B from near Beijing, China were completely sequenced and comparatively analyzed to investigate genetic stability and diversity of PlxyGV. PlxyGV-W, PlxyGV-B and PlxyGV-Wn consist of 100,941bp, 100,972bp and 100,999bp in length with G + C compositions of 40.71–40.73%, respectively, and share nucleotide sequence identities of 99.5–99.8%. The three individual isolates contain 118 putative protein-encoding ORFs in common. PlxyGV-W, PlxyGV-B and PlxyGV-Wn have ten, nineteen and six nonsynonymous intra isolate nucleotide polymorphisms (NPs) in six, fourteen and five ORFs, respectively, including homologs of five DNA replication/late expression factors and two per os infectivity factors. There are seventeen nonsynonymous inter isolate NPs in seven ORFs between PlxyGV-W and PlxyGV-B, seventy three nonsynonymous NPs in forty seven ORFs between PlxyGV-W and PlxyGV-Wn, seventy seven nonsynonymous NPs in forty six ORFs between PlxyGV-B and PlxyGV-Wn. Alignment of the genome sequences of nine PlxyGV isolates sequenced up to date shows that the sequence homogeneity between the genomes are over 99.4%, with the exception of the genome of PlxyGV-SA from South Africa, which shares a sequence identity of 98.6–98.7% with the other ones. No events of gene gain/loss or translocations were observed. These results suggest that PlxyGV genome is fairly stable in nature. In addition, the transcription start sites and polyadenylation sites of thirteen PlxyGV-specific ORFs, conserved in all PlxyGV isolates, were identified by RACE analysis using mRNAs purified from larvae infected by PlxyGV-Wn, proving the PlxyGV-specific ORFs are all genuine genes.

Parasitic Entamoeba spp. can infect many classes of vertebrates including humans and pigs. Entamoeba suis and zoonotic Entamoeba polecki have been identified in pigs, and swine are implicated as potential reservoirs for Entamoeba histolytica. However, the prevalence of Entamoeba spp. in pigs in southeastern China has not been reported. In this study, 668 fecal samples collected from 6 different regions in Fujian Province, southeastern China, were analyzed to identify three Entamoeba species by nested PCR and sequencing analysis. The overall prevalence of Entamoeba spp. was 55.4% (370/668; 95% CI 51.6% to 59.2%), and the infection rate of E. polecki ST1 was the highest (302/668; 45.2%, 95% CI 41.4% to 49.0%), followed by E. polecki ST3 (228/668; 34.1%, 95% CI 30.5% to 37.7%) and E. suis (87/668; 13.0%, 95% CI 10.5% to 15.6%). E. histolytica was not detected in any samples. Moreover, the coinfection rate of E. polecki ST1 and ST3 was 25.1% (168/668; 95% CI 21.9% to 28.4%), the coinfection rate of E. polecki ST1 and E. suis was 3.7% (25/668; 95% CI 2.3% to 5.2%), the coinfection rate of E. polecki ST3 and E. suis was 0.3% (2/668), and the coinfection rate of E. polecki ST1, E. polecki ST3, and E. suis was 4.0% (27/668; 95% CI 2.5% to 5.5%). A representative sequence (MK347346) was identical to the sequence of E. suis (DQ286372). Two subtype-specific sequences (MK357717 and MK347347) were almost identical to the sequences of E. polecki ST1 (FR686383) and ST3 (AJ566411), respectively. This is the first study to survey the occurrence and to conduct molecular identification of three Entamoeba species in southeastern China. This is the first report regarding mixed infections with E. suis, E. polecki ST1, and E. polecki ST3 in China. More research studies are needed to better understand the transmission and zoonotic potential of Entamoeba spp.

Marshallagia marshalli (Nematoda: Trichostrongylidae) infection can lead to serious parasitic gastroenteritis in sheep, goat, and wild ruminant, causing significant socioeconomic losses worldwide. Up to now, the study concerning the molecular biology of M. marshalli is limited. Herein, we sequenced the complete mitochondrial (mt) genome of M. marshalli and examined its phylogenetic relationship with selected members of the superfamily Trichostrongyloidea using Bayesian inference (BI) based on concatenated mt amino acid sequence datasets. The complete mt genome sequence of M. marshalli is 13,891 bp, including 12 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. All protein-coding genes are transcribed in the same direction. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes supported the monophylies of the families Haemonchidae, Molineidae, and Dictyocaulidae with strong statistical support, but rejected the monophyly of the family Trichostrongylidae. The determination of the complete mt genome sequence of M. marshalli provides novel genetic markers for studying the systematics, population genetics, and molecular epidemiology of M. marshalli and its congeners.

Toxoplasma gondii is a successful opportunistic parasite, affecting a wide range of vertebrate animals and humans. Genetic diversity of T. gondii in raccoon dogs (Nyctereutes procyonoides) is of great importance to understand the transmission of T. gondii in the environment. However, no information is available about the distribution of genetic diversity of T. gondii infection in raccoon dogs. This study was conducted to estimate the prevalence and genetic characterization of T. gondii from raccoon dogs in Shandong province, eastern China. A total of 314 brain tissue samples of raccoon dogs were collected and genomic DNA was extracted and assayed for T. gondii infection using semi-nested PCR targeting B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2(5'+3' SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) by multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn- PCR-RFLP) technology. Thirty-five (11.15%) of 314 DNA samples were detected positive. Only six samples were completely typed at all genetic loci, and these samples represented ToxoDB genotype#9. Two sample were typed at 9 genetic loci and one was typed at 8 genetic loci, all of them represented Type I. To our knowledge, this is the first report of genetic characterization of T. gondii in raccoon dogs in China. These results revealed the existence of genetic diversity of T. gondii in raccoon dogs in China. These data provided base-line information for controlling T. gondii infection in raccoon dogs.

Toxoplasma gondii, an obligate intracellular apicomplexan parasite, can infect homoiothermal vertebrate animals, including raccoon dogs (Nyctereutes procyonoides). Regretfully, data on T. gondii infection in raccoon dogs were limited in China. Therefore, to investigate the seroprevalence and to evaluate risk factors for T. gondii infection in raccoon dogs, a total of 1181 raccoon dog blood samples were collected from Jilin and Shandong provinces, China, from September to December 2014. The antibodies of T. gondii were examined using the modified agglutination test. Overall, the seroprevalence of T. gondii infection was 8.64% in the examined raccoon dogs. The prevalences of T. gondii infection were different among cities (ranging from 2.96% in Yantai to 17.62% in Qingdao), genders (female: 7.58%; male: 9.22%), and ages (young: 8.53%; subadult: 7.71%; adult: 7.73%). Region was considered as an important risk factor for T. gondii infection in this study. This is the first report of T. gondii infection in raccoon dogs in China, providing baseline information for prevention and control of T. gondii infection in raccoon dogs in Jilin and Shandong provinces, China.

Enterocytozoon bieneusi is an important opportunistic pathogen widely distributed in humans and animals that causes diarrhea or fatal diarrhea in immunocompromised hosts. To examine the infection status and molecular characteristics of E. bieneusi in pigs, 725 fecal samples were collected from pigs in six areas of Fujian Province. The E. bieneusi genotypes were identified based on the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene by nested PCR, and its population genetics were analyzed by multilocus sequence typing (MLST). The results showed that the infection rate of E. bieneusi was 24.4% (177/725), and 11 known genotypes (EbpC, EbpA, CHN-RR2, KIN-1, CHG7, CHS5, CM11, CHG23, G, PigEBITS, and D) and 2 novel genotypes (FJF and FJS) were identified. All the genotypes were found to be clustered into zoonotic Group 1. Moreover, 52 positive samples were successfully amplified at minisatellite and microsatellite loci and formed 48 distinct multilocus genotypes (MLGs). Further population structure analyses showed strong genetic linkage disequilibrium (LD) and several recombination events (Rm), indicating that E. bieneusi has a clonal population structure. This study is the first to investigate the prevalence and molecular characteristics of E. bieneusi in Fujian Province and could provide baseline data to control E. bieneusi infection in pigs and humans and deepen our understanding of the zoonotic risk of E. bieneusi and its distribution in China.

Background Little information about the prevalence of gastrointestinal parasites in yaks (Bos grunniens) in northwest China is available. Therefore, the objective of the study was to quantify faecal egg counts of gastrointestinal parasites (helminths and coccidia) in free-range yaks from Gannan Tibetan Autonomous Prefecture, Gansu Province, Northwest China. Results Parasites were detected in 290 of 733 (39.56%) faecal samples. The results showed that Strongylidae, Trichuris spp. and Eimeria spp. were detected all year round, Strongyloides papillosus was detected in autumn and summer, and Nematodirus spp. was detected in both autumn and spring. In contrast, Fasciola spp. was only detected in spring. The prevalence rates of parasitic infections in different seasons were significantly different. Conclusions To our knowledge, this is the first investigation of gastrointestinal parasites in yaks (Bos grunniens) in Gansu, China. The results demonstrated a high prevalence of gastrointestinal parasitic infections, specifically GN infections, in yaks in GTAP and these infections can cause economic losses to the local cattle industry.

Toxoplasma gondii can infect all the warm-blooded animals and humans and causes serious diseases especially in immuno-compromised patients and pregnant women. Rhoptry neck proteins (RONs) play an important role in the formation of moving junction, which mediates the invasion of this parasite. A recombinant plasmid pVAX-RON5p, which can express part of RON5 protein in the eukaryocyte, was generated and used to immune BALB/c mice for evaluating the protective efficacy against the acute and chronic infections of T. gondii. Both humoral and cellular immune responses were evoked in mice by pVAX-RON5p immunization, and a slightly prolonged survival period was detected in the immunized group (7.6 ± 3.31 days) compared to the blank control (4.9 ± 0.32 days) after acute T. gondii infection (P < 0.05). For chronic infection of T. gondii, the number of cysts in the brain of pVAX-RON5p-immunized mice decreased 25.8% compared to blank control (P < 0.05). Our data suggested that RON5p DNA vaccine can induce partial protective immunity against acute and chronic T. gondii infections.

Giardia intestinalis is a cosmopolitan protozoan parasite that can infect a range of animals, including dairy cattle. As information regarding the prevalence and genotyping of G. intestinalis infection in dairy cattle in northwestern China is limited, 2,945 feces samples from 1,224 dairy cattle in Gansu Province and from 1,614 in Ningxia Hui Autonomous Region (NXHAR) were examined between December 2012 and March 2014. The overall prevalence of G. intestinalis was 3.63% (107/2,945), with 2.63% and 4.38% in Gansu and NXHAR, respectively. Logistic regression analysis showed region, age and season to be significant risk factors for G. intestinalis infection. Assemblage analysis identified 106 assemblage E and one assemblage A at the triose phosphate isomerase (tpi) locus in this study. Intravariations were also detected at tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci within assemblage E, showing seven, three, and five new subtypes, respectively. Moreover, 13 new multilocus genotypes (E20-E32) were observed in assemblage E. Effective strategies and measures should be taken to prevent and control giardiasis in Gansu and NXHAR.

Giardia duodenalis is a common intestinal protozoa, which can cause the occurrence of diarrhea, weight loss, and even death in animals or human, this threatens the husbandry industry and public health. It can infect virtually humans and all domestic animals including sheep. Tan sheep is one of the most important sheep breeds, which is short-tailed indigenous sheep breed used for production of high quality meat and pelts in China. However, there are no report regarding the occurrence and multilocus genotyping of G. duodenalis in Tan sheep in northwestern China. Thus, the objective of the present study was to investigate the prevalence and multilocus genotypes of G. duodenalis in Tan sheep. 1014 fecal samples were collected from Tan sheep from Ningxia Hui Autonomous Region, and three loci (β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes) were amplified by nested PCR. The prevalence of G. duodenalis in Tan sheep was 14.5% (147/1014), two assemblages (assemblage A, n = 43; and E, n = 90) were detected, including one novel assemblage A at bg locus, one novel assemblage A at tpi locus, and 10 and 11 novel subtypes of assemblage E were detected at the bg and gdh loci, respectively. One MLGs was formed based on sequence variation among the three loci. Moreover, 9 Tan sheep were infected with two assemblages (A and E) based on the three loci. These findings expand the host range of G. duodenalis and revealed genetic diversity of G. duodenalis assemblages in Tan sheep.

Significance A majority of emerging infectious diseases in humans are transmitted from animals. It is generally agreed that our behavior can influence our exposure to such pathogens, but little is known regarding our role in shaping evolution in such pathogens. Such understanding would aid in their control, to the benefit of public health. Our results indicate that expansion of agriculture influenced not only the biogeography but also the virulence of Toxoplasma gondii. By linking landscape ecology to parasite virulence, our framework contributes a fundamentally unique perspective on the ecology and evolution of infectious disease., A majority of emerging infectious diseases in humans are zoonoses. Understanding factors that influence the emergence and transmission of zoonoses is pivotal for their prevention and control. Toxoplasma gondii is one of the most widespread zoonotic pathogens known today. Whereas only a few genotypes of T. gondii dominate in the Northern Hemisphere, many genotypes coexist in South America. Furthermore, T. gondii strains from South America are more likely to be virulent than those from the Northern Hemisphere. However, it is not clear what factor(s) shaped modern-day genetic diversity and virulence of T. gondii. Here, our analysis suggests that the rise and expansion of farming in the past 11,000 years established the domestic cat/mouse transmission cycle for T. gondii, which has undoubtedly played a significant role in the selection of certain linages of T. gondii. Our mathematical simulations showed that within the domestic transmission cycle, intermediately mouse-virulent T. gondii genotypes have an adaptive advantage and eventually become dominant due to a balance between lower host mortality and the ability to superinfect mice previously infected with a less virulent T. gondii strain. Our analysis of the global type II lineage of T. gondii suggests its Old World origin but recent expansion in North America, which is likely the consequence of global human migration and trading. These results have significant implications concerning transmission and evolution of zoonotic pathogens in the rapidly expanding anthropized environment demanded by rapid growth of the human population and intensive international trading at present and in the future.

Neospora caninum is an intracellular protozoan parasite which can cause abortion and stillbirth in ruminants. However, there is no information on Tibetan sheep N. caninum infection in China. A total of 2187 serum samples were collected from Tibetan sheep in the major production areas of Luqu, Maqu, and Tianzhu in Gansu province, and Nyingchi in southeast Tibet, China. All samples were analyzed for the presence of antibodies to N. caninum using a competitive-inhibition enzyme-linked immunoassay. Of the 2187 serum samples, 184 (8.4%, 95% CI 7.3-9.6) were tested N. caninum seropositive. The N. caninum seroprevalence ranged from 4.4% (95% CI 1.4–7.4) to 11.3% (95% CI 8.2–14.4) among different regions, seasons, ages, and pregnancies, and there was no statistical significance among those groups (P > 0.05). Seroprevalence in male (10.8% 69/638) (95% CI 8.4–13.2) was significantly higher than in female (7.4% 115/1549) (OR =1.51, 95% CI 6.1–8.7) (P < 0.01). To our knowledge, this is the first report of N. caninum seroprevalence in Tibetan sheep in China, which provides baseline data for the prevention and control of N. caninum infection in Tibetan sheep.

The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10?min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50?fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75?samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.

Toxoplasma gondii is a global pathogen that infects a wide range of animals and humans. During T. gondii infection, the spleen plays an important role in coordinating the adaptive and innate immune responses. However, there is little information regarding the changes in global gene expression within the spleen following T. gondii infection. To address this gap in knowledge, we examined the transcriptome of the mouse spleen following T. gondii infection. We observed differential expression of 2310 transcripts under these conditions. Analysis of KEGG and GO enrichment indicated that T. gondii alters multiple immune signaling cascades. Most of differentially expressed GO terms and pathways were downregulated, while immune-related GO terms and pathways were upregulated with response to T. gondii infection in mouse spleen. Most cytokines were upregulated in infected spleens, and all differentially expressed chemokines were upregulated which enhanced the immune cells chemotaxis to promote recruitment of immune cells, such as neutrophils, eosinophils, monocytes, dendritic cells, macrophages, NK cells, basophils, B cells, and T cells. Although IFN-γ-induced IDO (Ido1) was upregulated in the present study, it may not contribute a lot to the control of T. gondii because most differentially expressed genes involved in tryptophan metabolism pathway were downregulated. Innate immunity pathways, including cytosolic nucleic acid sensing pathway and C-type lectins-Syk-Card9 signaling pathways, were upregulated. We believe our study is the first comprehensive attempt to define the host transcriptional response to T. gondii infection in the mouse spleen.

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, which can infect warm-blooded animals and humans. The present study was performed to investigate the seroprevalence of T. gondii in horses in Xinjiang, northwestern China. A total of 637 blood samples were collected from seven regions in Changji Hui Autonomous Prefecture, Xinjiang in 2011 and assayed for T. gondiiantibodies using the modified agglutination test (MAT). Risk factors (age, gender, and region) related to seroprevalence were determined by a multivariate logistic regression analysis. A total of 200 horses (31.4%, 95% CI 27.79–35.00) were seropositive for T. gondii. Age, gender, and region present no association with seroprevalence (p>0.05) in the logistic regression analysis. The results indicated that T. gondii is widely prevalent in horses in Xinjiang, northwestern China, representing a serious threat to animal and human health. Therefore, more careful measures should be performed to control and prevent T. gondii infection in horses from Xinjiang, northwestern China. A toxoplasmose é uma zoonose global causada pelo Toxoplasma gondii, o qual pode infectar animais de sangue quente e seres humanos. Este estudo foi realizado com o objetivo de investigar a soroprevalência em cavalos para T. gondii, na região de Xinjiang, no Noroeste da China. Em 2011, foram recolhidas 637 amostras de sangue em sete distritos da Prefeitura Autônoma de Changji Hui do Xinjiang, as quais foram testadas para a presença de anticorpos, utilizando-se o teste de aglutinação modificado (MAT). Foram estimados fatores de risco relacionados com a soroprevalência (idade, sexo e distrito), através de uma análise de regressão logística multivariada. Um total de 200 equinos (31,4%, 95% IC 27,79 – 35,00) foi positivo para T. gondii. Idade, sexo e região estudada não apresentaram associação com a soroprevalência (p>0,05) na análise de regressão logística. Os resultados revelam que a infecção por T. gondii tem uma prevalência generalizada em todo o território de Xinjiang, no Noroeste da China, constituindo uma séria ameaça à saúde de animais e de humanos. Consequentemente, propõe-se que sejam adotadas medidas reforçadas para o controle e prevenção da infecção de cavalos por T. gondii, no Xinjiang, Noroeste da China.

Query (Q) fever is a vector-borne zoonosis caused by the obligate intracellular pathogen Coxiella burnetii. Animals, including dogs, cats, cattle, and sheep, can be infected by C. burnetii. However, little information is available about C. burnetii infection in Tibetan sheep in China. Antibodies against C. burnetii in Tibetan sheep serums, which were collected from Maqu County, Tianzhu County and Nyingchi Prefecture, China, between 2011 and 2013, were examined by enzyme-linked immunosorbent assay (ELISA). Overall, 14.39% (304/2112, 95% confidence interval [CI] 12.90-15.89) of the examined Tibetan sheep were positive for C. burnetii infection. The C. burnetii seroprevalence in male and female Tibetan sheep was 12.94% (95% CI 10.26-15.61) and 14.98% (95% CI 13.18-16.78), respectively. The seroprevalence varied from 13.19% (95% CI 10.08-16.30) to 14.96% (95% CI 12.31-17.62) in Tibetan sheep in different areas. In addition, the seroprevalence ranged from 13.16% (95% CI 9.83-16.50) to 15.10% (95% CI 13.15-17.04) in different age groups. The results of the present survey indicate that C. burnetii infection is highly prevalent in Tibetan sheep in China. This is the first report of C. burnetii infection in Tibetan sheep in China, extending the host range for C. burnetii. Further studies are warranted to understand better the epidemiological situation of C. burnetii transmission in these areas and elsewhere.

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, which can infect warm-blooded animals and humans. A serological survey was undertaken to examine the seroprevalence and risk factors associated with T. gondii infection in sika deer in northeastern China. 114 (13.46%, 95% CI 11.16-15.76) out of 847 serum samples were positive to T. gondii by modified agglutination test (MAT) at a 1:25 cut-off, with titers of 1:25 in 44, 1:50 in 32, 1:100 in 17, 1:500 in 11, 1:1500 or higher in 10. These samples were collected between November 2012 and October 2013 from Inner Mongolia, Jilin and Heilongjiang provinces in China. However, statistically significant differences were not observed between T. gondii seroprevalence and genders or regions of sika deer in the logistic regression analysis (P>0.05) and left out of the final model. Seroprevalence of T. gondii infection in male sika deer was 14.07% (95% CI 11.14-17.01), slightly higher than that in the female (12.38%) (95% CI 8.69-16.06) and seroprevalence of T. gondii infection in Harbin, Changchun city, Jilin city and Chifeng city were 12.02% (95% CI 7.60-16.44), 15.51% (95% CI 11.52-19.50), 12.27% (95% CI 7.23-17.31) and 12.50% (95% CI 7.38-17.63), respectively. Seasons of sampling were considered as main risk factors associated with T. gondii infection, autumn (15.32%) were more than two times (OR=1.98, 95% CI=1.18-3.33, P=0.01) at risk of acquiring T. gondii infection compared to winter (8.37%). Our results indicated a widespread exposure to T. gondii among sika deer in China. To our knowledge, this is the first report of T. gondii seroprevalence in sika deer in China.

Toxoplasma gondii is an obligatory intracellular protozoan, which infects most of the warm-blooded animals, causing serious public health problems and enormous economic losses worldwide. The rhoptry effector protein 54 (ROP54) has been indicated as a virulence factor that promotes Toxoplasma infection by modulating GBP2 loading onto parasite-containing vacuoles, which can modulate some aspects of the host immune response. In order to evaluate the immuno-protective value of ROP54, we constructed a eukaryotic recombinant plasmid expressing T. gondii ROP54 and intramuscularly immunized Kunming mice with this recombinant plasmid against acute and chronic toxoplasmosis. All mice immunized with pVAX-ROP54 elicited a high level of specific antibody responses, a significant increase of lymphocyte proliferation, and a significant level of Th1-type cytokines (IFN-γ, IL-2 and IL-12p70), in addition to an increased production of Th2-type cytokines (IL-4 and IL-10). These results demonstrated that pVAX-ROP54 induced significant cellular and humoral (Th1/Th2) immune responses, which extended the survival time (13.0±1.15days for pVAX-ROP54 vs 6.7±0.48days for pVAX I, 6.8±0.42days for PBS and 6.5±0.53 for blank control) and significantly reduced cyst burden (35.9% for pVAX-ROP54, 1% for pVAX I and 2% for PBS, compared with blank control) of immunized mice. These results indicate that the recombinant ROP54 plasmid can provide partial protection and might be a potential vaccine candidate against acute and chronic toxoplasmosis.

Enterocytozoon bieneusi, the most common zoonotic pathogen of microsporidiosis, has been found in various animals and humans, but no information is available concerning the prevalence and genotypes of E. bieneusi in white yaks (Bos grunniens). In the present study, 353 faecal samples from white yaks in Tianzhu Tibetan Autonomous County, Gansu Province, Northwestern China, were collected and examined by PCR amplification of the internal transcribed spacer gene to estimate E. bieneusi prevalence and identify their genotypes. Of the 353 faecal samples, 4 (1.13%) were tested E. bieneusi-positive. Sequences analysis revealed that two known genotypes, namely, I (n = 1) and BEB4 (n = 2), and a novel genotype, namely, WCY1 (n = 1), were found in this study. Among them, genotype WCY1 was clustered into Group 1, and genotypes I and BEB4 belonged to Group 2. The present study firstly indicates the existence of E. bieneusi in yaks in Gansu Province, Northwestern China. This is also the first record of E. bieneusi in white yaks. Effective measures should be taken to control E. bieneusi infection in white yaks, other animals, and humans.

From January to July 2012, we looked for evidence of subclinical A (H1N1) pdm09 and seasonal human influenza viruses infections in healthy dogs in China. Sera from a total of 1920 dogs were collected from Guangdong, Guangxi, Fujian and Jiangxi provinces. We also examined archived sera from 66 dogs and cats that were collected during 2008 from these provinces. Using hemagglutination inhibition (HI) and microneutralization (MN) assays, we found that only the dogs sampled in 2012 had elevated antibodies (≥ 1:32) against A(H1N1)pdm09 virus and seasonal human influenza viruses: Of the 1920 dog sera, 20.5 % (n = 393) had elevated antibodies against influenza A(H1N1) pdm09 by the HI assay, 1.1 % (n = 22), and 4.7 % (n = 91) of the 1920 dogs sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus by the HI assay. Compared with dogs that were raised on farms, dogs that were raised as pets were more likely to have elevated antibodies against A(H1N1)pdm09 and seasonal human influenza viruses. Seropositivity was highest among pet dogs, which likely had more diverse and frequent exposures to humans than farm dogs. These findings will help us better understand which influenza A viruses are present in dogs and will contribute to the prevention and control of influenza A virus. Moreover, further in-depth study is necessary for us to understand what roles dogs play in the ecology of influenza A.

The synergistic protective efficacy of murine interleukin 21 (mIL-21) and mIL-15 administrated with DNA vaccine against acute and chronic Toxoplasma gondii infection in mice was investigated using T. gondii MIC8 (TgMIC8) as a model. We cloned mIL-21 and mIL-15 from splenic tissues of Kunming mice, and constructed eukaryotic plasmid pVAX/mIL-15, pVAX/mIL-21, and pVAX/mIL-21/mIL-15, respectively. After immunizing with pVAX/TgMIC8 in the presence or absence of these cytokines, immune responses were analyzed using lymphoproliferative assay, cytokine and serum antibody measurements, flow cytometric surface markers on lymphocytes and protection against acute and chronic T. gondii infection. Mice receiving pVAX/TgMIC8 alone developed a strong humoral responses and Th1 type cellular immune responses, and showed an increase of CD4+ and CD8+ T cells compared with all the controls. Adding pVAX/mIL-21 to pVAX/TgMIC8 compared to pVAX/TgMIC8 resulted in only a slight increase in humoral and cellular immune responses, and this immune response was lower than that induced by the pVAX/mIL-15 combined with pVAX/TgMIC8. Co-administration of pVAX/mIL-21/mIL-15 combined with pVAX/TgMIC8 elicited the strongest humoral and cellular immune responses among all the groups, leading to significantly increased survival time against acute infection and the significant reduction of tissue cysts, compared to all the controls. Synergy of mIL-21 and mIL-15 can facilitate specific humoral as well as cellular immune responses elicited by DNA vaccine against acute and chronic T. gondii infection in mice.

Toxoplasma gondii infection is a serious health problem of humans and animals worldwide. T. gondii eukaryotic initiation factor-2α (TgIF2α) plays a crucial role in parasite viability and is an important virulence factor of T. gondii. To evaluate the vaccine potential of TgIF2α, we constructed a novel eukaryotic plasmid pVAX-IF2α expressing TgIF2α from the RH strain and validated expression and immunogenicity in vitro in the Marc145 cell expression system by indirect immunofluorescence (IFA). Administration of pVAX-IF2α intramuscularly induced specific humoral immune responses including high levels of specific TgIF2α IgG antibody and a mixed IgG1/IgG2a response with a predominance of IgG2a production. The cellular immune response was elicited, showing significant production of IFN-γ and IL-2 associated with Th1 type response, and thus strong cell-mediated cytotoxic activity with increased frequencies of IFN-γ parameters analyzed in both CD4(+) and CD8(+) T cell compartments (CD4(+) IFN-γ(+) T cells and CD8(+) IFN-γ(+) T cells). Immunization resulted in partial protection against acute and chronic toxoplamosis in outbred Kunming mice, demonstrated by a significantly prolonged survival time (15.9±4.6 days) after challenge with the virulent RH strain and significant reduction in brain cysts (44.1%) against chronic infection with PRU cyst in contrast to control mice. Our data suggested that pVAX-IF2α could be used as a DNA vaccine candidate against both acute and chronic T. gondii infection by the activation of effective humoral and cellular immune responses.

The objective of this study was to determine the seroprevalence, risk factors and genotypes of Toxoplasma gondii in white yaks (Bos grunniens) in China. A total of 974 serum samples were collected from white yaks in Tianzhu Tibetan Autonomous County (TTAC), Gansu province, northwest China for detecting T. gondii antibodies by the modified agglutination test (MAT), and 414 tissues belonging to 138 white yaks were collected for detecting T. gondii DNA by amplification of B1 gene with a semi-nested PCR. A total of 155 serum samples (15.91%) were seropositive for T. gondii antibodies at a 1:100 cut-off, and 10 DNA samples (7.25%) were positive for the T. gondii B1 gene, which were genetically characterized using multilocus PCR-RFLP. Only one genotype (ToxoDB#9) was identified from two samples with complete genotyping results. Statistically significant differences were not observed between T. gondii seroprevalence and gender, season or pregnancy in the logistic regression analysis (P>0.05). Ages of white yaks was considered as a main risk factor associated with T. gondii infection. Our results indicated a widespread exposure to T. gondii among white yaks, and revealed the genotype ToxoDB#9 of T. gondii in white yaks for the first time in China.

The prevalence of Toxoplasma gondii infection in birds has epidemiological significance because birds are indeed considered as a good indicator of environmental contamination by T. gondii oocysts. In this study, the prevalence of T. gondii in 313 house sparrows in Lanzhou, northwestern China was assayed by the modified agglutination test (MAT). Antibodies to T. gondii were positive in 39 (12.46%) of 313 samples (MAT titer ≥ 1:5). Tissues of heart, brain, and lung from the 39 seropositive house sparrows were tested for T. gondii DNA, 11 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 9 genetic markers, including 8 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8 and an apicoplast locus Apico. Of them, 4 isolates were genotyped with complete data for all loci, and 2 genotypes (Type II variants; ToxoDB #3 and a new genotype) were identified. These results showed that there is a potential risk for human infection with T. gondii in this region. To our knowledge, this is the first report of T. gondii seroprevalence in house sparrows in China.

Toxoplasma gondii is a significant zoonotic parasite which can cause congenital infection and abortion in warm-blooded animals and humans. Microneme protein 13 (MIC13) plays an important role in attachment and penetration of the host cell by T. gondii. In this study, a DNA vaccine expressing mic13 of T. gondii was constructed and its protective efficacy was evaluated in Kunming L615(H2k) mice. Immunization with pVAX-TgMIC13 induced a strong immune responses demonstrated by significant lymphocyte proliferation, cytokine production and antibody responses. Immunized mice showed increased survival time (21.3±11.3 days) and reduced number of cysts in brain of mice (57.14%) after challenge with tachyzoites of the virulent T. gondii RH strain and cysts of the T. gondii PRU strain, respectively, demonstrating that T. gondii MIC13 is a potential vaccine candidate, worth being included in future vaccine development against acute and chronic T. gondii infection.

Ticks are blood-sucking ectoparasites of great medical and veterinary significance that can transmit bacteria, protozoa, fungi and viruses, and cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genome of Rhipicephalus sanguineus from China (RSC) and compared with that of R. sanguineus from USA (RSU). Nucleotide sequence difference in the full mt genome was 11.23% between RSC and RSU. For the 13 protein-coding genes, comparison revealed sequence divergences at both the nucleotide (9.34-15.65%) and amino acid (2.54-19.23%) levels between RSC and RSU. In addition, sequence comparison of the conserved mt cox1 and cytb genes among multiple individual R. sanguineus revealed substantial nucleotide differences between RSC and RSU but limited sequence variation within RSC. Phylogenetic analysis of ticks based on the amino acid sequence data of 13 protein-coding genes revealed that R. sanguineus from China and R. sanguineus from USA represent sister taxa (likely separate species). Taken together, the findings support the recently proposal that R. sanguineus tick may represents a species complex of at least two closely related species.

The seroprevalence of Chlamydiaceae infection in Tibetan pigs in Tibet, China, was examined by indirect hemagglutination assay (IHA), between April, 2010, and December, 2010. A total of 71 of 427 serum samples (16.63%, 95% confidence interval [CI] 15.31–17.95] were positive for Chlamydiaceae antibodies. Forty Chlamydiaceae seropositives from 232 samples were recorded in sera from Nyingchi (17.24%, 95% CI 15.40–19.08) and 31 positives were recorded in 195 serum samples from Mainling (15.90%, 95% CI 14.02–17.78). The investigation showed that the prevalence in female animals was 17.61% (95% CI 15.22–20.00), and in male animals it was 12.72% (95% CI 11.07–14.37). The prevalence ranged from 0% to 20.61% (95% CI 17.81–23.48) among different age groups, with a higher prevalence in growing pigs (p

Dicrocoelium chinensis is commonly known as the "lancet fluke" or "small liver fluke" which lives in the bile ducts and gall bladder of domestic and wild ruminants. In this study, we examined sequence variability in two mitochondrial (mt) genes, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes, among D. chinensis isolated from domestic yaks in Gansu and Sichuan provinces, China. A portion of the cox1 (pcox1) and nad1 (pnad1) genes was amplified by PCR separately from the individual adult D. chinensis, and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of pcox1 and pnad1 were 358 and 458 bp, respectively. The A+T contents of the sequences were 58.94-59.22% for pcox1 and 60.04-60.48% for pnad1, respectively. The intra-specific sequence variations within D. chinensis were 0-0.84% for pcox1 and 0-0.87% for pnad1. The inter-specific sequence differences among members of the examined trematodes were significantly higher, being 22.9-29.3% and 33.6-41.5% for pcox1 and pnad1, respectively. Phylogenetic analysis utilizing combined sequences of pcox1 and pnad1 using Bayesian inference revealed that all of the D. chinensis isolates grouped together with high statistical support. These findings demonstrated the existence of low intra-specific variation in mitochondrial DNA (mtDNA) sequences among the D. chinensis isolates from domestic yaks in China and had implications for studying the molecular epidemiology and population genetics of D. chinensis.

Bluetongue (BT), caused by bluetongue virus (BTV), is an arthropod-borne viral disease in ruminants. However, information about BTV infection in yaks in China is limited. Moreover, no such data concerning BTV in Tibetan sheep is available. Therefore, 3771 serum samples were collected from 2187 Tibetan sheep and 1584 yaks between April 2013 and March 2014 from Tibetan Plateau, western China, and tested for BTV antibodies using a commercially available ELISA kit. The overall seroprevalence of BTV was 17.34% (654/3771), with 20.3% (443/2187) in Tibetan sheep and 13.3% (211/1584) in yaks. In the Tibetan sheep group, the seroprevalence of BTV in Luqu, Maqu, Tianzhu, and Nyingchi Prefecture was 20.3%, 20.8%, 20.5%, and 19.1%, respectively. The seroprevalence of BTV in different season groups varied from 16.5% to 23.4%. In the yak group, BTV seroprevalence was 12.6%, 15.5%, and 11.0% in Tianzhu, Maqu, and Luqu counties, respectively. The seroprevalence in different seasons was 12.6%, 15.5%, 15.4%, and 9.0% in spring, summer, autumn, and winter, respectively. The season was the major risk factor concerning BTV infection in yaks (P<0.05). The date of the BTV seroprevalence in Tibetan sheep and yaks provides baseline information for controlling BT in ruminants in western China.

Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1 % N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.

Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

Better understanding of the molecular changes associated with disease is essential for identifying new routes to improved therapeutics and diagnostic tests. The aim of this study was to investigate the dynamic changes in the metabolic profile of mouse sera during T. gondii infection. We carried out untargeted metabolomic analysis of sera collected from female BALB/c mice experimentally infected with the T. gondii Pru strain (Genotype II). Serum samples were collected at 7, 14 and 21 day post infection (DPI) from infected and control mice and were subjected to liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based global metabolomics analysis. Multivariate statistical analysis identified 79 differentially expressed metabolites in ESI+ mode and 74 in ESI− mode in sera of T. gondii-infected mice compared to the control mice. Further principal component analysis (PCA) and partial least squares-discrimination analysis (PLS-DA) identified 19 dysregulated metabolites (5 in ESI+ mode and 14 in ESI− mode) related to the metabolism of amino acids and energy metabolism. The potential utility of these metabolites as diagnostic biomarkers was validated through receiver operating characteristic (ROC) curve analysis. These findings provide putative metabolite biomarkers for future study and allow for hypothesis generation about the pathophysiology of toxoplasmosis.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is an important pathogen of cattle worldwide, causing reproductive disorders in adult cattle and mucosal disease in calves. However, limited information about BVDV infection in yaks (Bos grunniens) in China is available, especially in white yaks which is a unique yak breed that only lives in Tianzhu Tibetan Autonomous County (TTAC), Gansu Province, northwest China. Therefore, we conducted a cross-sectional study to estimate the seroprevalence and risk factors associated with BVDV infection in 1584 yaks in Gansu province, northwest China, between April 2013 and March 2014 using an indirect ELISA test. The overall seroprevalence of BVDV in yaks was 37.56 % (595/1584), with 45.08 % (275/610) in black yaks and 32.85 % (320/974) in white yaks. Moreover, positive yaks were found in all four regions, varied from 33.22 to 40.31 %. Male yaks had a similar seroprevalence (37.84 %) with that of the female yaks (37.11 %). Season, species and geographical origins of yaks were considered as risk factors analyzed by logistic regression model. To our knowledge, this is the first report of seroprevalence and risk factors associated with BVDV infection in white yaks in China.

Enzootic bovine leukosis (EBL) is a chronic lymphosarcoma disease of cattle caused by bovine leukemia virus (BLV). No information is available concerning the epidemiology of BLV infection in yaks (Bos mutus). One thousand five hundred and eighty-four serum samples from 610 black yaks and 974 white yaks from Gansu province, northwest China, were collected between April 2013 and March 2014 and tested for BLV antibodies using a commercially available ELISA kit. The overall BLV seroprevalence in yaks was 21.09% (334/1584), with 24.26% (148/610) black yaks and 19.10% (186/974) white yaks yielding positive results. Risk factor analysis indicated that with the exception of breed (OR = 1.36, 95% CI = 1.06–1.73,P<0.05), the age, region, gender, farm, and the numbers of pregnancies were not considered as risk factors for the presence of BLV in yaks included in this study. This is the first report of BLV infection in yaks in China, which provides information for controlling BLV infection in yaks.

This study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 5 (nad5) and cytochrome b (cytb), among Trichuris ovis isolates from different hosts in Guangdong Province, China. A portion of the cox1 (pcox1), nad5 (pnad5) and cytb (pcytb) genes was amplified separately from individual whipworms by PCR, and was subjected to sequencing from both directions. The size of the sequences of pcox1, pnad5 and pcytb was 618, 240 and 464 bp, respectively. Although the intra-specific sequence variations within T. ovis were 0-0.8% for pcox1, 0-0.8% for pnad5 and 0-1.9% for pcytb, the inter-specific sequence differences among members of the genus Trichuris were significantly higher, being 24.3-26.5% for pcox1, 33.7-56.4% for pnad5 and 24.8-26.1% for pcytb, respectively. Phylogenetic analyses using combined sequences of pcox1, pnad5 and pcytb, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), indicated that all of the T. ovis isolates grouped together with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA sequences among T. ovis isolates from different hosts, and have implications for studying molecular epidemiology and population genetics of T. ovis.

Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA) sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp) than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI) and maximum likelihood (ML)] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.

Host cell invasion byToxoplasma gondiiis tightly related to microneme protein 6 (MIC6) andT. gondiiperforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts ofT. gondiistrain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels ofToxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P< 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P> 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.

In the present study, we sequenced the complete mt genome (14,022 bp) of parasitic nematode Contracaecum rudolphii B and its structure and organization compared with Anisakis simplex s.l. The mt genome of C. rudolphii B is slightly longer than that of A. simplex s.l. (13,916 bp). C. rudolphii B mt genome is circular, and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. This genome contains a high A+T (70.5%) content. The mt gene order for C. rudolphii B is the same as those for A. simplex s.l., but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of C. rudolphii B are TTG and ATT. Six protein-coding genes use TAA as a stop codon whereas five genes use T and one genes use TAG as a termination codon. This pattern of codon usage reflects the strong bias for A and T in the mt genome of C. rudolphii B. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (Bayes, ML and MP), all revealed distinct groups with high statistical support, indicating that C. rudolphii B and A. simplex s.l. is distinct but closely related species. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the C. rudolphii B, and should have implications for the molecular diagnosis, prevention and control of anisakidosis in humans and animals.

Toxoplasma gondiiwith worldwide distribution has received substantial medical and scientific attentions as it causes serious clinical and veterinary problems especially for pregnant women and immunocompromised patients. Heat shock protein 40 (HSP40) plays a variety of essential roles in the pathogenesis of this protozoan parasite. In order to detail the genetic diversity of HSP40 gene, 16T. gondiistrains from different hosts and geographical locations were used in this study. Our results showed that HSP40 sequence of the examined strains was between 6621 bp and 6644 bp in length, and their A+T content was from 48.54% to 48.80%. Furthermore, sequence analysis presented 195 nucleotide mutation positions (0.12%–1.14%) including 29 positions in CDS (0.02%–0.12%) compared withT. gondiiME49 strain (ToxoDB: TGME49_265310). Phylogenetic assay revealed thatT. gondiistrains representing three classical genotypes (Types I, II, and III) were completely separated into different clusters by maximum parsimony (MP) method, but Type II and ToxoDB#9 strains were grouped into the same cluster. These results suggested that HSP40 gene is not a suitable marker forT. gondiipopulation genetic research, though three classical genotypes ofT. gondiicould be differentiated by restriction enzymesMscI andEarI existing in amplicon C.

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting mammals and birds including humans. Rhoptry protein 18 has been implicated as an important virulence factor. In this study, we constructed a DNA vaccine expressing rhoptry protein 18 (ROP18) of T. gondii, and evaluated the immune response and protective immunity in Kunming mice. The gene sequence encoding ROP18 was inserted into the eukaryotic expression vector pVAX I. Intramuscular immunization of mice with pVAX-ROP18 elicited specific humoral responses and stimulated lymphoproliferation (P0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P0.05). By the expression of the CD69, an activation marker of CD4+ and CD8+ T cells, we found that pVAX-ROP18 enhanced the activation of CD4+ and CD8+ T cells in lymphoid in mice. After lethal challenge, the mice immunized with the pVAX-ROP18 showed a significantly increased survival time (27.9±15.1 days) compared with control mice which died within 7 days of challenge (P0.05). Our results show for the first time, that a ROP18 vaccine construct can enhance the T. gondii-specific CTL. Th1 responses and increased survival suggested that ROP18 is a promising vaccine candidate against infection with T. gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which can invade and multiply within the macrophages of humans and most warm-blooded animals. Macrophages are important effector cells for the control and killing of intracellular T. gondii, and they may also serve as long-term host cells for the replication and survival of the parasite. In the present study, we explored the proteomic profile of macrophages of the specific pathogen-free Kunming mice at 24 h after infection with tachyzoites of the virulent T. gondii RH strain using two-dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight (TOF)/TOF tandem mass spectrometry. Totally, 60 differentially expressed protein spots were identified. Among them, 52 spots corresponded to 38 proteins matching to proteins of the mouse, including actin, enolase, calumenin, vimentin, plastin 2, annexin A1, cathepsin S, arginase-1, arachidonate 12-lipoxygenase, and aminoacylase-1. Functional prediction using Gene Ontology database showed that these proteins were mainly involved in metabolism, structure, protein fate, and immune responses. The findings provided an insight into the interactive relationship between T. gondii and the host macrophages, and will shed new lights on the understanding of molecular mechanisms of T. gondii pathogenesis.

Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli , a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly ( P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a severe disease of pigs, causing significant economic losses to the pig industry worldwide, including the tropical and subtropical regions. In order to obtain the baseline prevalence of M. hyopneumoniae in pigs from intensive farms in southern China, double-sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect M. hyoneumoniae antibodies in 460 pig serum samples collected from 12 administrative cities in China's southern Guangdong province. According to the proportions of the infected animals, among the 12 intensive farms, only two of them showed no infection of M. hyoneumoniae and the seroprevalence ranged from 0% to 90%, with an averaged prevalence of 45.7%. The highest prevalence was found in breeding boars (68.8%), followed by sows (54.5%). These data showed that the infection of pigs with M. hyopneumoniae is severe, and boars might be more important carriers and transfers of M. hyoneumoniae than sows. Integrated strategies and measures should be taken to control the infection of pigs with M. hyopneumoniae in southern China.

Microneme protein 8 (MIC8) is considered a new essential invasion factor in Toxoplasma gondii. In the present study, a deoxyribonucleic acid vaccine expressing MIC8 of T. gondii was constructed and the immune response it induced in Kunming mice was evaluated. The gene sequence encoding MIC8 was inserted into the eukaryotic expression vector pVAX I, and the pVAX-MIC8 expression plasmid was constructed, and the plasmid diluted with PBS to 100 mg/100 microl was injected into the Kunming mice muscularly. Levels of IgG antibody, gamma-interferon (IFN-gamma), interleukin-2 (IL-2), interleukin-4, and interleukin-10 were detected. The mice were challenged with tachyzoites of the virulent T. gondii RH strain at the 14th day after the last immunization to observe the survival time. The high level of IFN-gamma, IL-2, and IgG antibody indicated that mice vaccinated with recombinant pVAX-MIC8 plasmid could elicit strong cellular and humoral immune responses and showed a significantly increased survival time (10.3 +/- 0.9 days) compared with control mice which died within 5 days of challenge infection. These data demonstrate that the T. gondii MIC8 is a potential vaccine candidate against toxoplasmosis.

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Microneme proteins which are responsible for adhesion and invasion have been implicated as vaccine candidates. In this study, we constructed a DNA vaccine expressing microneme protein 6 (MIC6) of T. gondii, and evaluated the immune response it induced in Kunming mice. The gene sequence encoding MIC6 was inserted into the eukaryotic expression vector pVAXI. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the group immunized with pVAX-MIC6 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong lymphoproliferative response, and significant levels of IFN-gamma, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or phosphate-buffered saline, respectively. These results demonstrate that pVAX-MIC6 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-MIC6 showed an increased survival time (13.3+/-1.2 days) compared with control mice died within 7 days of challenge. Our data demonstrate, for the first time, that MIC6 triggered a strong humoral and cellular response against T. gondii, and that the antigen is a potential vaccine candidate against toxoplasmosis, worth further development.

© 2016 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the down regulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets.

Toxoplasma gondii is an obligate, intracellular protozoan that can infect virtually all warm-blooded animals. Humans can be infected through the consumption of undercooked meat of farm animals. However, there are few reports on pathogenic mechanism and immune regulation during the interaction between T. gondii and domesticated animals. RNA extracted from pig peripheral blood mononuclear cells (PBMCs) infected with T. gondii RH strain at different time points (0 h, 8 h, 24 h, and 48 h) were reverse transcribed into cDNA and sequenced using Illumina technology. After ruling out short-length and low-quality sequences, more than 18 million clean reads and 14 million unique reads were acquired in each group. Clean reads were then mapped to the pig reference genome and differently expressed (DE) genes were identified. DE genes increased along with the extension of infection time. For function classification and pathway analysis, annotated DE genes were categorized into three Gene Ontology (GO) categories and assigned to six major Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, 10 DE genes were identified to be involved in some important immune processes and signaling pathways at the early infection stage. This is the first transcriptomic analysis of pig PBMCs infected by T. gondii. Functional DE genes and KEGG pathways indentified at different time points in this study will help to better understand the host immunity and defense mechanisms and promote the prevention and control of T. gondii infection.