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Taenia pisiformis is one of the most important parasites of canines and rabbits. T. pisiformis cysticercus (the larval stage) causes severe damage to rabbit breeding, which results in huge economic losses. In this study, the genetic variation of T. pisiformis was determined in Sichuan Province, China. Fragments of the mitochondrial cytochrome b (cytb) (922 bp) gene were amplified in 53 isolates from 8 regions of T. pisiformis. Overall, 12 haplotypes were found in these 53 cytb sequences. Molecular genetic variations showed 98.4% genetic variation derived from intra-region. FST and Nm values suggested that 53 isolates were not genetically differentiated and had low levels of genetic diversity. Neutrality indices of the cytb sequences showed the evolution of T. pisiformis followed a neutral mode. Phylogenetic analysis revealed no correlation between phylogeny and geographic distribution. These findings indicate that 53 isolates of T. pisiformis keep a low genetic variation, which provide useful knowledge for monitoring changes in parasite populations for future control strategies.

Roundworms of the genus Baylisascaris are the most common parasitic nematodes of the intestinal tracts of wild mammals, and most of them have significant impacts in veterinary and public health. Mitochondrial (mt) genomes provide a foundation for studying epidemiology and ecology of these parasites and therefore may be used to assist in the control of Baylisascariasis. Here, we determined the complete sequences of mtDNAs for Baylisascaris schroederi, Baylisascaris ailuri and Baylisascaris transfuga, with 14,778 bp, 14,657 bp and 14,898 bp in size, respectively. Each mtDNA encodes 12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs, typical for other chromadorean nematodes. The gene arrangements for the three Baylisascaris species are the same as those of the Ascaridata species, but radically different from those of the Spirurida species. Phylogenetic analysis based on concatenated amino acid sequences of 12 protein-coding genes from nine nematode species indicated that the three Baylisascaris species are more closely related to Ascaris suum than to the three Toxocara species (Toxocara canis, Toxocara cati and Toxocara malaysiensis) and Anisakis simplex, and that B. ailuri is more closely related to B. transfuga than to B. schroeder. The determination of the complete mt genome sequences for these three Baylisascaris species (the first members of the genus Baylisascaris ever sequenced) is of importance in refining the phylogenetic relationships within the order Ascaridida, and provides new molecular data for population genetic, systematic, epidemiological and ecological studies of parasitic nematodes of socio-economic importance in wildlife.

Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 μg/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis.

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.

To analyse genetic variability and population structure, 84 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from various host species at different sites of the Tibetan plateau in China were sequenced for the whole mitochondrial nad1 (894 bp) and atp6 (513 bp) genes. The vast majority were classified as G1 genotype (n=82), and two samples from human patients in Sichuan province were identified as G3 genotype. Based on the concatenated sequences of nad1+atp6, 28 different haplotypes (NA1-NA28) were identified. A parsimonious network of the concatenated sequence haplotypes showed star-like features in the overall population, with NA1 as the major haplotype in the population networks. By AMOVA it was shown that variation of E. granulosus within the overall population was the main pattern of the total genetic variability. Neutrality indexes of the concatenated sequence (nad1+atp6) were computed by Tajima's D and Fu's Fs tests and showed high negative values for E. granulosus, indicating significant deviations from neutrality. FST and Nm values suggested that the populations were not genetically differentiated.

Taenia multiceps (Cestoda: Taeniidae), a worldwide cestode parasite, is emerging as an important helminthic zoonosis due to serious or fatal central nervous system disease commonly known as coenurosis in domestic and wild ruminants including humans. Herein, a fatty acid-binding protein (FABP) gene was identified from transcriptomic data in T. multiceps. This gene, which contains a complete coding sequence, was amplified by reverse-transcriptase polymerase chain reaction. The corresponding protein, which was named TmFABP, had a molecular weight of 14 kDa, and subsequently was recombinantly expressed in Escherichia coli. The fusion protein was purified on Ni-NTA beads (Bio-Rad). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses showed that the purified recombinant protein caused immunogenicity. Immunohistochemical studies showed that TmFABP was expressed at the tegumental level in the protoscolices and in the cells between the body wall and parenchyma layer of the cestode. In sections from gravid proglottids, intense staining was detected in the uterus and eggs. Based on this, TmFABP could be switched on during differentiation of germinative layers to protoscoleces and from metacestodes to adult worms. Taken together, our results already reported for T. multiceps suggest the possibility of TmFABP developing a vaccine to control and prevent coenurosis.

The comparative efficacy of 2 anthelmintics (ivermectin and levamisole) against Baylisascaris transfuga migrating and encapsulated larvae was studied in mice. A total of 60 BALB/c mice inoculated each with about 1,000 embryonated B. transfuga eggs were equally divided into 6 groups (A-F) randomly. Mice of groups A and B were treated with ivermectin and levamisole, respectively, on day 3 post-infection (PI). Mice of groups A-C were killed on day 13 PI. Similarly, groups D and E were treated with ivermectin and levamisole, respectively, on day 14 PI, and all mice of groups D-F were treated on day 24 PI. The groups C and F were controls. Microexamination was conducted to count the larvae recovering from each mouse. The percentages of reduction in the number of migrating larvae recovered from group A (ivermectin) and B (levamisole) were 88.3% and 81.1%, respectively. In addition, the reduction in encapsulated larvae counts achieved by ivermectin (group D) and levamisole (group E) was 75.0% and 49.2%, respectively. The results suggested that, to a certain extent, both anthelmintics appeared to be more effective against migrating larvae than encapsulated larvae. However, in the incipient stage of infection, ivermectin may be more competent than levamisole as a larvicidal drug for B. transfuga.

BACKGROUND: The heartworm Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis in dogs and cats, and also infects a wide range of wild mammals as well as humans. One bottleneck for the design of fundamentally new intervention and management strategies against D. immitis may be the currently limited knowledge of fundamental molecular aspects of D. immitis. METHODOLOGY/PRINCIPAL FINDINGS: A next-generation sequencing platform combining computational approaches was employed to assess a global view of the heartworm transcriptome. A total of 20,810 unigenes (mean length = 1,270 bp) were assembled from 22.3 million clean reads. From these, 15,698 coding sequences (CDS) were inferred, and about 85% of the unigenes had orthologs/homologs in public databases. Comparative transcriptomic study uncovered 4,157 filarial-specific genes as well as 3,795 genes potentially involved in filarial-Wolbachia symbiosis. In addition, the potential intestine transcriptome of D. immitis (1,101 genes) was mined for the first time, which might help to discover 'hidden antigens'. CONCLUSIONS/SIGNIFICANCE: This study provides novel insights into the transcriptome of D. immitis and sheds light on its molecular processes and survival mechanisms. Furthermore, it provides a platform to discover new vaccine candidates and potential targets for new drugs against dirofilariosis.