Prevalence and molecular characterization of Trichomonas gallinae from pigeons in Anhui, China.

Trichomonas gallinae, a protozoan parasite causing avian trichomonosis, exhibits a widespread global prevalence. It primarily affects the upper digestive tract of birds and has resulted in significant ecological problems worldwide. This study aimed to investigate the prevalence and genotypes of T. gallinae in Anhui Province, China. A total of 1612 oropharyngeal swab samples were collected from pigeon farms in Anhui Province to determine the prevalence of T. gallinae infection. The results revealed 565 (35.1%) positive samples of T. gallinae. Significant differences in infection rates were observed among different regions and age groups. Furthermore, the ITS1/5.8 S/ITS2 region was amplified, sequenced, and subjected to phylogenetic analysis. Genotypes A and B of T. gallinae were identified, and genotype B was the dominant genotype in Anhui Province. This is the first report on the prevalence and molecular characterization of T. gallinae in Anhui Province, China. Additionally, we integrated reports on the prevalence and genotype of T. gallinae in relevant provinces in China.

APDDD: Animal parasitic diseases and drugs database.

Animal parasitic diseases not only have an economic impact, but also have serious social and public health impacts. Although antiparasitic drugs can treat these diseases, it seems difficult for users to comprehensively utilize the information, due to incomplete and difficult data collection. Thus, there is an urgent need to establish a comprehensive database, that includes parasitic diseases and related drugs. In this paper, we develop a knowledge database dedicated to collecting and analyzing animal parasitic diseases and related drugs, named Animal Parasitic Diseases and Drugs Database (APDDD). The current version of APDDD includes animal parasitic disease data of 8 major parasite classifications that cause common parasitic diseases and 96 subclass samples mined from many literature and authoritative books, as well as 182 antiparasitic drugs. Furthermore, we utilized APDDD data to add a knowledge graph representing the relationships between parasitic diseases, drugs, and the targeted gene of drugs acting on parasites. We hope that APDDD will become a good database for animal parasitic diseases and antiparasitic drugs research and that users can gain a more intuitive understanding of the relationships between parasitic diseases, drugs, and targeted genes through the knowledge graph.

Epidemiological survey of tick-borne pathogens in dogs in Anhui Province, China.

Tick-borne diseases have continued to increase worldwide in both developing and many developed countries due to the widespread of different tick species and tick's adaptability to different climatic weather. In order to investigate the prevalence of the tick-borne pathogens, EDTA-anticoagulated whole blood samples were aseptically collected from 765 pet dogs in twenty veterinary clinics located in sixteen prefecture-level cities in Anhui Province, China, and the samples were examined and analyzed for tick-borne pathogens using both microscopy and PCR. Our result analysis revealed 17(2.22%) positive samples to Babesia spp and 4(0.52%) positive samples to Hepatozoon spp, of which case of co-infection was recorded in Lu'An and Chuzhou. The BLAST analysis results of the 18S rRNA gene revealed that the dogs were infected with Babesia gibsoni and Hepatozoon canis. All samples were negative for Anaplasma spp., Ehrlichia spp., and Rickettsia spp. This is the first molecular report of B. gibsoni and H. canis in dogs in Anhui, China.

Development and validation of a machine learning algorithm prediction for dense granule proteins in Apicomplexa.

BACKGROUND: Apicomplexa consist of numerous pathogenic parasitic protistan genera that invade host cells and reside and replicate within the parasitophorous vacuole (PV). Through this interface, the parasite exchanges nutrients and affects transport and immune modulation. During the intracellular life-cycle, the specialized secretory organelles of the parasite secrete an array of proteins, among which dense granule proteins (GRAs) play a major role in the modification of the PV. Despite this important role of GRAs, a large number of potential GRAs remain unidentified in Apicomplexa. METHODS: A multi-view attention graph convolutional network (MVA-GCN) prediction model with multiple features was constructed using a combination of machine learning and genomic datasets, and the prediction was performed on selected Neospora caninum protein data. The candidate GRAs were verified by a CRISPR/Cas9 gene editing system, and the complete NcGRA64(a,b) gene knockout strain was constructed and the phenotypes of the mutant were analyzed. RESULTS: The MVA-GCN prediction model was used to screen N. caninum candidate GRAs, and two novel GRAs (NcGRA64a and NcGRA64b) were verified by gene endogenous tagging. Knockout of complete genes of NcGRA64(a,b) in N. caninum did not affect the parasite's growth and replication in vitro and virulence in vivo. CONCLUSIONS: Our study showcases the utility of the MVA-GCN deep learning model for mining Apicomplexa GRAs in genomic datasets, and the prediction model also has certain potential in mining other functional proteins of apicomplexan parasites.

Development and application of an indirect ELISA to detect antibodies to Neospora caninum in cattle based on a chimeric protein rSRS2-SAG1-GRA7.

Neospora caninum is an important apicomplexan parasite causing neosporosis in cattle. The disease is recognized as one of the most important cause of reproductive problems and abortion in cattle worldwide. In this context, we developed an indirect enzyme-linked immunosorbent assays (ELISA) with chimeric protein rSRS2-SAG1-GRA7 to diagnose antibodies to Neospora-infection. This indirect ELISA was compared to indirect fluorescent antibody test (IFAT) and western blotting (WB), and the sensitivity and specificity results of ELISA were calculated to be 86.7 and 96.1%, respectively. The overall coincidence rate was 92.6% using IFAT and WB. Additionally, 329 aborting dairy cattle serum samples were tested using this ELISA to evaluate the prevalence of N. caninum in Ningxia, China. The positive rate of N. caninum in these farms was from 19.05 to 57.89%, and the mean rate was 41.64% (±11.01%), indicating that infection with N. caninum may be one of the important causes of cattle abortion in this region. This established rSRS2-SAG1-GRA7 indirect ELISA is capable for detecting the antibodies against N. caninum, and it could be a useful screening tool for monitoring the epidemiology of neosporosis in cattle.

The mitogenome of Ophidascaris wangi isolated from snakes in China.

Different species of the genus Ophidascaris (Baylis, 1921; Nematoda: Ascaridida, Ascaridoidea) are intestinal parasites of various snake species. More than 30 Ophidascaris species have been reported worldwide; however, few molecular genetic studies have been conducted on this genus. We sequenced the complete mitogenome of Ophidascaris wangi parasitizing two snake species of the family Colubridae, i.e., Elaphe carinata (Günther, 1864) and Dinodon rufozonatum. The mitogenome sequence of O. wangi was approximately 14,660 base pairs (bp) long and encoded 36 genes, including 12 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, and 22 transfer RNA genes. Gene arrangement, genome content, and transcription direction were in line with those in Toxascaris leonina (Linstow, 1902; Ascaridida: Ascarididae). Phylogenetics of O. wangi and other ascaridoids were reconstructed based on the concatenated amino acid sequences of 12 PCGs, and on nucleotide sequences of 12 PCGs and two rRNA genes. Phylogenetic analyses were performed using maximum likelihood and Bayesian inference methods, and the results suggested that O. wangi constitutes a sister clade of Ascaris, Parascaris, Baylisascaris, and Toxascaris within the family Ascarididae, which is a sister clade of Toxocaridae. The mitogenome sequence of O. wangi obtained from the present study will be useful for future identification of the nematode worms in the genus Ophidascaris and will increase the understanding of population genetics, molecular epidemiology, and phylogenetics of ascaridoid nematodes in snakes.

A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides.

Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.

A serpin from the tick Rhipicephalus haemaphysaloides: Involvement in vitellogenesis.

Tick serpins are involved in enzyme activity, food digestion, blood-feeding, immune response and anticoagulation. Little is known about the potential roles of serpins in tick reproduction. RHS8, a serpin from the tick Rhipicephalus haemaphysaloides, has an open reading frame 1212 bp long and encodes a protein that has 404 amino acids and a predicted molecular weight of 45 kDa. RHS8 exhibits 89.58 % amino acid identity with RmS15 in Rhipicephalus microplus. RHS8 was expressed primarily in larvae and nymphs. RHS8 mRNA expression in the ovaries, fat bodies and salivary glands were up-regulated from feeding to ovipositing ticks. RNAi results showed that RHS8 dsRNA-injected ticks had a lower body weight, longer feeding time, fewer eggs laid and lower egg hatchability. Tick reproduction, such as egg laying and hatching, was disrupted by RNAi. Compared with the control group, ovaries of the RHS8 interference group were light brown color, indicating a reduction in yolk granule accumulation. Western blot results showed that the expression of RHVg3 and RHVg4 proteins in ovaries was reduced in the RHS8 dsRNA-injected group. These results indicate that RHS8 is related to tick reproduction and its interference affects vitellogenesis.

Molecular detection and genotype distribution of Enterocytozoon bieneusi in farmed silver foxes (Vulpes vulpes) and arctic foxes (Vulpes lagopus) in Shandong Province, eastern China.

Enterocytozoon bieneusi is an opportunistic enteric pathogen which can infect a wide range of animal species and humans. It is the most diagnosed species of Microsporidia in humans and has an impact on public health. Many infected animals including foxes may be a potential source for transmitting E. bieneusi to humans. However, limited information is available on the E. bieneusi prevalence and genotypes in farmed foxes in China. Therefore, in the present study, 344 fresh fecal samples were collected from farmed foxes (Vulpes vulpes and Vulpes lagopus) in Shandong Province, and the prevalence and genotypes of E. bieneusi were examined based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 9% (31/344); of them, 6.5% (9/138) in farmed silver foxes (V. vulpes) and 10.7% (22/206) in farmed arctic foxes (V. lagopus). Moreover, four known (Hum-q1, NCF2, HND-1, and Type IV) and two novel E. bieneusi genotypes (SDF1 and SDF2) were identified in farmed foxes in the present study. All of the E. bieneusi genotypes belonged to the zoonotic group based on phylogenetic analysis. In addition, 2, 4, 0, and 11 samples were successfully amplified at MS1, MS3, MS4, and MS7 loci, respectively. The present study reveals E. bieneusi prevalence and genotype distribution in farmed foxes in Shandong Province and enlarged the host and geographic information of E. bieneusi in China.

Evaluation of immune protection against Toxoplasma gondii infection in mice induced by a multi-antigenic DNA vaccine containing TgGRA24, TgGRA25 and TgMIC6.

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In this study, recombinant eukaryotic expression plasmids (pVAX-GRA24, pVAX-GRA25 and pVAX-MIC6) were constructed, and then injected into Kunming mice intramuscularly, as cocktailed plasmids or as single-gene plasmids. We evaluated immune protective responses by detecting the titer of antibodies and cytokine production of IFN-γ, IL-2, IL-4, IL-10, IL-12 and IL-23, the percentages of the subclasses of T lymphocytes, as well as the records of the survival time and cyst decrement in the brain of the mouse model after challenge with the T. gondii RH and Pru strains, respectively. Compared with the control groups, antibody and cytokine production were significantly increased, while the survival times of mice in all immunized groups were also prolonged, and the number of T. gondii cysts in their brains were decreased significantly (29.03% for pVAX-GRA24; 40.88% for pVAX-GRA25; 37.70% for pVAX-MIC6; 48.06% for pVAX-GRA24 + pVAX-GRA25; and 55.37% for pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). The mouse group immunized with the three-gene cocktail (TgGRA24 + TgGRA25 + TgMIC6) had better performance in each detection index than the mouse groups immunized with the two-gene cocktail of TgGRA24 + TgGRA25, which was better than that in the group immunized with the single gene vaccine of TgGRA24, TgMIC6 or TgGRA25. In conclusion, TgGRA24 or TgGRA25 may be good vaccine candidates against T. gondii infection, but the three-gene cocktail of TgGRA24, TgMIC6 and TgGRA25 may induce the strongest protective immunity. Further studies of multi-antigenic DNA vaccines or cocktailed vaccines against T. gondii infection are necessary.

TITLE: Évaluation de la protection immunitaire contre l'infection par Toxoplasma gondii chez la souris, induite par un vaccin à ADN multi-antigénique contenant TgGRA24, TgGRA25 et TgMIC6. ABSTRACT: L'infection à Toxoplasma gondii est répandue chez les humains et les animaux dans le monde entier. Dans cette étude, des plasmides d'expression eucaryotes recombinants (pVAX-GRA24, pVAX-GRA25 et pVAX-MIC6) ont été construits, puis injectés à des souris Kunming par voie intramusculaire, comme cocktails de plasmides ou comme plasmides à un seul gène. Nous avons évalué les réponses de protection immunitaire en détectant le titre des anticorps et la production de cytokines IFN-γ, IL-2, IL-4, IL-10, IL-12 et IL-23, les pourcentages des sous-classes des lymphocytes T, ainsi qu'en mesurant les temps de survie et de décrément des kystes dans le cerveau du modèle souris après challenge par les souches RH et Pru de T. gondii, respectivement. Comparativement aux groupes témoins, la production d'anticorps et de cytokines était significativement accrue, tandis que le temps de survie des souris de tous les groupes immunisés était également prolongé et que le nombre de kystes de T. gondii dans leur cerveau diminuait de manière significative (29,03 % pour pVAX-GRA24 ; 40,88 % pour pVAX-GRA25 ; 37,70 % pour pVAX-MIC6 ; 48,06 % pour pVAX-GRA24 + pVAX-GRA25 ; 55,37 % pour pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). Le groupe de souris immunisées avec les cocktails à trois gènes (TgGRA24 + TgGRA25 + TgMIC6) présentait la meilleure performance dans chaque indice de détection par rapport aux groupes de souris immunisées avec des cocktails à deux gènes de TgGRA24 + TgGRA25, qui était supérieur à ceux immunisés avec les vaccins monogéniques TgGRA24, TgMIC6 ou TgGRA25. En conclusion, TgGRA24 ou TgGRA25 peuvent être de bons candidats au vaccin contre l'infection à T. gondii, mais un cocktail à trois gènes de TgGRA24, TgMIC6 et TgGRA25 peut induire la plus forte immunité protectrice. Des études supplémentaires sur les vaccins à ADN multi-antigéniques ou les vaccins en cocktail contre l'infection à T. gondii sont nécessaires.

A low-cost, automated parasite diagnostic system via a portable, robotic microscope and deep learning.

Manual hand counting of parasites in fecal samples requires costly components and substantial expertise, limiting its use in resource-constrained settings and encouraging overuse of prophylactic medication. To address this issue, a cost-effective, automated parasite diagnostic system that does not require special sample preparation or a trained user was developed. It is composed of an inexpensive (~US$350), portable, robotic microscope that can scan over the size of an entire McMaster chamber (100 mm2 ) and capture high-resolution (~1 µm lateral resolution) bright field images without need for user intervention. Fecal samples prepared using the McMaster flotation method were imaged, with the imaging region comprising the entire McMaster chamber. These images are then automatically segmented and analyzed using a trained convolution neural network (CNN) to robustly separate eggs from background debris. Simple postprocessing of the CNN output yields both egg species and egg counts. The system was validated by comparing accuracy with hand-counts by a trained operator, with excellent performance. As a further demonstration of utility, the system was used to conveniently quantify drug response over time in a single animal, showing residual disease due to Anthelmintic resistance after 2 weeks.

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets.

Caused by porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PED) is an acute infectious disease which causes damage to the intestine including intestinal villus atrophy and shedding, leading to serious economic losses to the pig industry worldwide. In order to obtain detailed information about the pathogenesis and host immune response in a PEDV-infected host for first In vivo study we used high-throughput sequencing to analyze the gene expression differences of the small intestinal mucosa after infection with PEDV. Transcripts obtained were over 65,525,000 clean reads after reassembly were 22,605 genes detected, of which 22,248 were known genes and 371 new genes were predicted. Moreover, 3168 genes expression was up-regulated and 3876 genes down-regulated. (Gene Ontology) GO annotation and functional enrichment analysis indicated that all of the DEGs (differentially expressed genes) were annotated into biological process, cellular component and molecular function. Most of these unigenes are annotated in cellular processes, the cell and binding. KEGG analysis of the DEGs showed that a total of 7044 DEGs unigenes were annotated into 323 pathways classified into 6 main categories. Most of these unigenes are annotated were related to immune system response to the infectious diseases pathways. In addition, 20 DEGs were verified by quantitative real-time PCR. As the first, in vivo, RNAseq analysis of piglets and PEDV infection, our study provides knowledge about the transcriptomics of intestinal mucosa in PEDV-infected piglets, from which a complex molecular pathways and pathogenesis-related biological processes are involved in PEDV interaction with piglet intestinal mucosa.

[Toxoplasma gondii ROP38 promotes the maturation of dendritic cells mediated by TLR4].

Objective To investigate the effect of rhoptry protein 38 (ROP38) from Toxoplasma gondii (T. gondii) on the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4) induction in vitro. Methods The total RNA from T. gondii RH strain was extracted by guanidine thiocyanate method, and then cDNA was synthesized with reverse transcription reaction. After ROP38 gene was amplified by PCR, the recombinant pGEX-4T-ROP38 was constructed and expressed under IPTG induction. The recombinant ROP38 protein was detected by SDS-PAGE and Western blot analysis. The secondary structure and antigenicity of the ROP38 were predicted through DNAStar8.0 and ProtScale. In vitro, DCs were isolated and cultured for 6 days, then reacted with ROP38 antigen for 2 hours. The CD11c was detected by flow cytometry, and the data were analyzed with ANOVA by SPSS 23.0 software. Results The amplified gene was about 516 bp as expected. The sequence analysis showed that its homology was 99% compared with the reported sequence (XM_002366710.2) from GenBank. It was found that the relative molecular mass (Mr) of recombinant ROP38 was 45 kD. The prediction of structure indicated that there were 5 α-helices, 5 ß-sheets, 5 hydrophilic regions and 8 potential epitopes in ROP38 protein. In vitro, the expression of CD11c on DCs was significantly up-regulated after stimulated with ROP38, and the expression level of CD11c in ROP38 infection group was significantly higher than that of the other groups. Conclusion ROP38 promotes the maturation of DCs mediated by TLR4.

A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings.

In this paper we report the development of a cost-effective, modular, open source, and fully automated slide-scanning microscope, composed entirely of easily available off-the-shelf parts, and capable of bright field and fluorescence modes. The automated X-Y stage is composed of two low-cost micrometer stages coupled to stepper motors operated in open-loop mode. The microscope is composed of a low-cost CMOS sensor and low-cost board lenses placed in a 4f configuration. The system has approximately 1 micron resolution, limited by the f/# of available board lenses. The microscope is compact, measuring just 25×25×30 cm, and has an absolute positioning accuracy of ±1 µm in the X and Y directions. A Z-stage enables autofocusing and imaging over large fields of view even on non-planar samples, and custom software enables automatic determination of sample boundaries and image mosaicking. We demonstrate the utility of our device through imaging of fluorescent- and transmission-dye stained blood and fecal smears containing human and animal parasites, as well as several prepared tissue samples. These results demonstrate image quality comparable to high-end commercial microscopes at a cost of less than US$400 for a bright-field system, with an extra US$100 needed for the fluorescence module.

INVESTIGATION ON CRYPTOSPORIDIUM INFECTIONS IN WILD ANIMALS IN A ZOO IN ANHUI PROVINCE.

To assess Cryptosporidium infections among wild animals in a zoo located in Anhui province, we conducted an investigation on the fecal samples collected from 44 primates, 41 herbivores, 44 carnivores and omnivores, and 103 birds in the zoo with the use of Sheather's sugar flotation technique and modified acid-fast staining. Cryptosporidium oocysts were detected in the fecal samples from six primates, two herbivores, four carnivores and omnivores, and seven birds by using Sheather's sugar flotation technique; the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 13.64, 4.88, 9.09, and 6.80%, respectively. Modified acid-fast staining detected the presence of Cryptosporidium oocysts in the fecal samples of one primate, three herbivores, 0 carnivores and omnivores, and one bird, and the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 2.27, 7.32, 0.00, and 0.97%, respectively. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) and phylogenetic analysis with the use of the neighbor-joining (NJ) method based on the aligned partial small-subunit (SSU) rRNA gene sequences showed that the protozoan pathogen isolated from primates was Cryptosporidium hominis and the pathogen isolated from camels ( Camelus dromedarius ) was Cryptosporidium andersoni. Subtyping the Cryptosporidium hominis by 60-kDa glycoprotein (GP60) gene phylogenetic analysis showed the Cryptosporidium hominis belongs to the subtype IdA and IbA.

MIC16 gene represents a potential novel genetic marker for population genetic studies of Toxoplasma gondii.

BACKGROUND: The zoonotic agent Toxoplasma gondii is distributed world-wide, and can infect a broad range of hosts including humans. Microneme protein 16 of T. gondii (TgMIC16) is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. However, little is known of the TgMIC16 sequence diversity among T. gondii isolates from different hosts and geographical locations. RESULTS: In this study, we examined sequence variation in MIC16 gene among T. gondii isolates from different hosts and geographical regions. The entire genomic region of the MIC16 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the MIC16 gene sequences. The results of sequence alignments showed two lengths of the sequence of MIC16 gene among all the examined 12 T. gondii strains: 4391 bp for strains TgCatBr5 and MAS, and 4394 bp for strains RH, TgPLH, GT1, PRU, QHO, PTG, PYS, GJS, CTG and TgToucan. Their A+T content ranged from 50.30 to 50.59 %. A total of 107 variable nucleotide positions (0.1-0.9 %) were identified, including 29 variations in 10 exons and 78 variations in 9 introns. Phylogenetic analysis of MIC16 sequences showed that typical genotypes (Type I, II and III) were able to be grouped into their respective genotypes. Moreover, the three major clonal lineages (Type I, II and III) can be differentiated by PCR-RFLP using restriction enzyme Pst I. CONCLUSIONS: Phylogenetic analysis and PCR-RFLP of the MIC16 locus among T. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages (Type I, II and III) into their respective genotypes, suggesting that MIC16 gene may provide a novel potential genetic marker for population genetic studies of T. gondii isolates.

The complete mitochondrial genomes of Gnathostoma doloresi from China and Japan.

Gnathostoma doloresi is one of the neglected pathogens causing gnathostomiasis. Although this zoonotic parasite leads to significant socioeconomic concerns globally, little is known of its genetics and systematics. In the present study, we sequenced and characterized the complete mitochondrial (mt) genomes of G. doloresi isolates from China and Japan. The lengths of the mt genomes of the G. doloresi China and Japan isolates are 13,809 and 13,812 bp, respectively. Both mt genomes encode 36 genes, including 12 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. The gene order, transcription direction, and genome content are identical with its congener G. spinigerum. Phylogenetic analyses based on concatenated amino acid sequences of 12 PCGs by Bayesian inference (BI) indicated that G. doloresi are closely related to G. spinigerum. Our data provide an invaluable resource for studying the molecular epidemiology, phylogenetics, and population genetics of Gnathostoma spp. and should have implications for further studies of the diagnosis, prevention, and control of gnathostomiasis in humans and animals.

[Morphological Characteristics and Molecular Identification of Trichuris sp. from Giraffe].

Objective: To investigate the morphological characteristics of Trichuris sp. from giraffe in Hefei wild zoo and identify its species using molecular techniques. Methods: Morphological characteristics of Trichuris collected from giraffe were analyzed. The internal transcribed spacer 1（ITS-1） was amplified by PCR and the PCR product was sequenced. The resulting sequence was homology analysis in GenBank and its heredity evolution tree was constructed by MEGA 4.0 software. Results: The male worms had a body length of 35.89-58.56 mm, an esophagus to body length ration of 0.29-0.40, and a spicule length of 1.96-3.89 mm. The thick and thin proportions of body were 7.02-23.45 mm and 28.05-40.05 mm respectively. These data showed different degrees of variation with previous reports. The PCR resulted in a product of 491 bp, comprising part of 18S rRNA and full length ITS-1. Sequence alignment showed that the identified Trichuris was most homologous（98.6%） with T. bos taurus HE608848, T. capreolus JX218218, and T. japanese AB367795, but it was only 46.0% homologous with T. discolor AB367794. In the heredity evolution tree, it was not located on the same branch as T. discolor, T. ovis and T. bos taurus. Conclusions: The Trichuris sp. collected from giraffe is different from previous reports in morphology and ITS-1 sequence. Further research is needed to determine if it is a new species.

[Establishment of LAMP Detection for Toxoplasma gondii Based on the 529 bp Repeat Sequence].

Objective: To establish the loop mediated isothermal amplification（LAMP） technique for determining Toxoplama gondii. Methods: The primers for LAMP of the conserved 529 bp sequence was designed by the Primer Explorer 4.0 software. The LAMP reaction was made on the constructed pMD-19T-529 bp recombinant plasmid as a template, and was optimized in loop primer, concentrations of betaine and MgSO4, and reaction temperature. The optimized LAMP and PCR were performed in ultra-pure water, on pig genome, Cryptosporidium parvum genome, Isospora suis genome, and pMD-19T-529 bp, respectively, to test the sensitivity of LAMP. The pMD-19T-529 bp was serially diluted to 109-100 copies/ml to test the specificity of LAMP. Results: LAMP using the designed primers amplified the 529 bp fragment of T. gondii. The optimized LAMP system had a total reaction volume of 25 µl, with the optimal concentrations of betaine and MgSO4 being 0.4 mol/L and 6 mmol/L, respectively. The amplification efficiency of 30 min reaction in the presence of loop primer was comparable to that of 60 min reaction without loop primer, which indicated that addition of loop primer shortened the reaction time by 30 min. The optimal reaction condition was 63 ℃ 30 min, 80 ℃ 3 min. The established LAMP method specifically amplifed the 529 bp fragment of T. gondii, and its efficiency was 10 times of PCR（detection threshold 103 copies/ml vs. 104 copies/ml）. Conclusion: The established LAMP for detecting Toxoplama gondii 529 bp repeat sequence shows high specificity and sensitivity.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene.

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.