Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercus tenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Genetic diversity of Echinococcus granulosus sensu lato in China: Epidemiological studies and systematic review.

Cystic echinococcosis (CE) is a neglected tropical zoonosis caused by Echinococcus granulosus sensu lato (s.l.) and remains a major public health concern globally. Here, CE isolates (n = 3310) with clearly defined genotypes and geographical origins in China were retrieved through our epidemiological survey (2016-2020) and systematic review (1992-2020). Existing known genotypes of Echinococcus granulosus sensu lato (E. granulosus s.l.) except for G4 have been found in China, particularly on the Tibetan Plateau, where their genetic diversity is unique to that part of the world. According to the systematic review, genetic compositions of E. granulosus s.l. in China were as follows: E. granulosus (G1, G3), 98.3%; Echinococcus ortleppi (G5), 0.1%; Echinococcus intermedius (G6, G7), 1.4%; and Echinococcus canadensis (G8, G10), 0.2%. Specifically, G1 was responsible for 97.7% of infections and characterized by the broadest host ranges and geographic distributions. Our epidemiological results showed a relatively stable genetic composition of E. granulosus s.l. in sheep and yaks from three CE hyperendemic provinces (Xinjiang, Sichuan, Qinghai). A higher proportion of fertile cysts were found in sheep (287/406, 70.7%) than in yaks (28/184, 15.2%). During the past 29 years, 51 cox1 haplotypes of E. granulosus s.l. were endemic in China. The ancestral haplotype (Hap_2) remained the most common haplotype, 12 relatively common haplotypes were endemic and nine newly reported haplotypes were found during the survey. Overall, our results demonstrate that the compulsory immunization of sheep and the pilot EG95 vaccination campaign in yaks are well matched with the current genotypic situation. In addition to yaks, we advocate for more surveillance of CE isolates from pigs, cattle, goats and camels, since their roles in the transmission and reservation of E. granulosus s.l. have been largely ignored in China.

Transcriptome-based analysis of putative allergens of Chorioptes texanus.

BACKGROUND: Mites of the genus Chorioptes are non-burrowing and cause mange in a wide range of domestic and wild animals including cattle, horses, sheep, goats, panda, moose, camelids, mydaus and alpacas. Molecular biology and host-parasite interactions of Chorioptes texanus are poorly understood, and only a few C. texanus genes and transcript sequences are available in public databases including the allergen genes. METHODS: Chorioptes texanus RNA was isolated from mites, and the transcriptome of C. texanus was analyzed using bioinformatics tools. Chorioptes texanus unigenes were compared with the allergen protein sequences from the mite allergen database website to predict the potential allergens. Chorioptes texanus putative allergen unigenes were compared with hydrolase genes by building a C. texanus hydrolase gene library with the best match of the homologous sequences. Three allergen genes were cloned and expressed, their recombinant proteins were purified and their allergenic activities were preliminarily investigated. RESULTS: Transcriptome sequencing (RNA-Seq) of C. texanus was analyzed and results demonstrated that 33,138 unigenes were assembled with an average length of 751 bp. A total of 15,130 unigenes were annotated and 5598 unigenes were enriched in 262 KEGG signaling pathways. We obtained 209 putative allergen genes and 34 putative allergen-hydrolase genes. Three recombinant allergen proteins were observed to induce different degrees of allergic reactions on rabbit skin. CONCLUSIONS: The present transcriptome data provide a useful basis for understanding the host-parasite interaction and molecular biology of the C. texanus mite. The allergenic activities of recombinant Euroglyphus maynei 1-like (Eur m 1-like) protein, Dermatophagoides ptreronyssinus 1-like (Der p 1-like) protein and Dermatophagoides ptreronyssinus 7-like (Der p 7-like) protein were preliminarily investigated by intradermal skin test. Meanwhile, differences in eosinophil counts were observed in different injected sites of the skin. The identification of putative allergen genes and hydrolase genes offers opportunities for the development of new diagnostic, prevention and treatment methods.

A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto, Echinococcus multilocularis and Echinococcus canadensis in China.

BACKGROUND: Echinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency. METHODS: In the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method. RESULTS: The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/µl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/µl, 10 pg/µl and 5 pg/µl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms. CONCLUSIONS: The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.

Genetic variation in mitochondrial cox2 of Heterakis gallinarum from poultry in Sichuan, China.

Heterakis gallinarum is one of the common parasitic nematodes found in the caecum of poultry. To investigate the genetic diversity and genetic structure of the H. gallinarum population in Sichuan, we amplified and sequenced the complete mitochondrial (mt) cytochrome c oxidase subunit II (cox2) gene of 59 H. gallinarum isolates from seven different geographical regions, then analyzed their genetic polymorphisms. All cox2 genes of the 59 H. gallinarum isolates were 696 bp in length, with an average A + T content of 67.1%. Fifty-nine sequences contained 34 variable sites, and were classified into 23 haplotypes (HS1-HS23). The values of haplotype diversity (Hd) and nucleotide diversity (π) were 0.688 and 0.00288, respectively. Based on values of FST and Nm (FST = 0.01929, Nm = 12.71), there was a frequent gene flow but no significant genetic differentiation observed among the populations. The network map showed that the most prominent haplotype was HS1, and the other haplotypes (HS2-HS23) were centered on HS1 with a star-like topology, indicating that H. gallinarum had previously experienced a population expansion. To our knowledge, this is the first research on the population genetics of H. gallinarum based on mitochondrial cox2.

Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages.

BACKGROUND: Psoroptic mange is a chronic, refractory, contagious and infectious disease mainly caused by the mange mite Psoroptes ovis, which can infect horses, sheep, buffaloes, rabbits, other domestic animals, deer, wild camels, foxes, minks, lemurs, alpacas, elks and other wild animals. Features of the disease include intense pruritus and dermatitis, depilation and hyperkeratosis, which ultimately result in emaciation or death caused by secondary bacterial infections. The infestation is usually transmitted by close contact between animals. Psoroptic mange is widespread in the world. In this paper, the transcriptome of P. ovis is described following sequencing and analysis of transcripts from samples of larvae (i.e. the Pso_L group) and nymphs and adults (i.e. the Pso_N_A group). The study describes differentially expressed genes (DEGs) and genes encoding allergens, which help understanding the biology of P. ovis and lay foundations for the development of vaccine antigens and drug target screening. METHODS: The transcriptome of P. ovis was assembled and analyzed using bioinformatic tools. The unigenes of P. ovis from each developmental stage and the unigenes differentially between developmental stages were compared with allergen protein sequences contained in the allergen database website to predict potential allergens. RESULTS: We identified 38,836 unigenes, whose mean length was 825 bp. On the basis of sequence similarity with seven databases, a total of 17,366 unigenes were annotated. A total of 1,316 DEGs were identified, including 496 upregulated and 820 downregulated in the Pso_L group compared with the Pso_N_A group. We predicted 205 allergens genes in the two developmental stages similar to genes from other mites and ticks, of these, 14 were among the upregulated DEGs and 26 among the downregulated DEGs. CONCLUSION: This study provides a reference transcriptome of P. ovis in absence of a reference genome. The analysis of DEGs and putative allergen genes may lay the foundation for studies of functional genomics, immunity and gene expression profiles of this parasitic mite species.

Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha.

Heterakis gallinae and Heterakis beramporia are the most prevalent nematode infecting native chicken breed, causing major economic losses. In the present study, the complete mitochondrial genomes (mt) of H. gallinae and H. beramporia were amplified by long-PCR and then sequenced. The complete mt genomes of H. gallinae and H. beramporia were 13,973bp and 14,012bp in size, respectively. Both mt genomes contain 12 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. All genes are transcribed in the same direction and the gene arrangement is identical to Ascaridia spp. Phylogenetic analysis based on the 12 protein-coding genes revealed that the family Heterakidae (represented by H. gallinae and H. beramporia) was more closely related to the infraorder Ascaridomorpha than it was to the infraorder Oxyuridomorpha. The present study determined the complete mt genome sequences for two Heterakis species, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these Heterakis parasites.

Absence of population genetic structure in Heterakis gallinarum of chicken from Sichuan, inferred from mitochondrial cytochrome c oxidase subunit I gene.

Population genetics information provides a foundation for understanding the transmission and epidemiology of parasite and, therefore, may be used to assist in the control of parasitosis. However, limited available sequence information in Heterakis gallinarum has greatly impeded the study in this area. In this study, we first investigated the genetic variability and genetic structure of H. gallinarum. The 1325 bp fragments of the mitochondrial COX1 gene were amplified in 56 isolates of H. gallinarum from seven different geographical regions in Sichuan province, China. The 56 sequences were classified into 22 haplotypes (H1-H22). The values of haplotype diversity (0.712) and nucleotide diversity (0.00158) in Sichuan population indicate a rapid expansion occurred from a relatively small, short-term effective population in the past. The haplotype network formed a distribution around H1 in a star-like topology, and the haplotypes did not cluster according to their geographical location. Similar conclusions could be made from MP phylogenetic tree. The Fst value (Fst<0.16965) and AMOVA analysis revealed that no significant genetic differentiation was observed among the seven different geographical populations. Neutrality tests (Tajima's D and Fu's Fs) and mismatch analysis indicated that H. gallinarum experienced a population expansion in the past. Our results indicated that H. gallinarum experienced a rapid population expansion in the past, and there was a low genetic diversity and an absence of population structure across the population.

The complete mitochondrial genome of the scab mite Psoroptes cuniculi (Arthropoda: Arachnida) provides insights into Acari phylogeny.

BACKGROUND: Limited available sequence information has greatly impeded population genetics, phylogenetics and systematics studies in the subclass Acari (mites and ticks). Mitochondrial (mt) DNA is well known to provide genetic markers for investigations in these areas, but complete mt genomic data have been lacking for many Acari species. Herein, we present the complete mt genome of the scab mite Psoroptes cuniculi. METHODS: P. cuniculi was collected from a naturally infected New Zealand white rabbit from China and identified by morphological criteria. The complete mt genome of P. cuniculi was amplified by PCR and then sequenced. The relationships of this scab mite with selected members of the Acari were assessed by phylogenetic analysis of concatenated amino acid sequence datasets by Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP). RESULTS: This mt genome (14,247 bp) is circular and consists of 37 genes, including 13 genes for proteins, 22 genes for tRNA, 2 genes for rRNA. The gene arrangement in mt genome of P. cuniculi is the same as those of Dermatophagoides farinae (Pyroglyphidae) and Aleuroglyphus ovatus (Acaridae), but distinct from those of Steganacarus magnus (Steganacaridae) and Panonychus citri (Tetranychidae). Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes, with three different computational algorithms (BI, ML and MP), showed the division of subclass Acari into two superorders, supported the monophylies of the both superorders Parasitiformes and Acariformes; and the three orders Ixodida and Mesostigmata and Astigmata, but rejected the monophyly of the order Prostigmata. CONCLUSIONS: The mt genome of P. cuniculi represents the first mt genome of any member of the family Psoroptidae. Analysis of mt genome sequences in the present study has provided new insights into the phylogenetic relationships among several major lineages of Acari species.

Isolation and identification of acaricidal compounds in Eupatorium adenophorum petroleum ether extract and determination of their acaricidal activity against Psoroptes cuniculi.

We used multiple silica gel column chromatography and thin-layer chromatography coupled with (1)H nuclear magnetic resonance (NMR) and (13)C NMR to separate and identify the active acaricidal ingredients in Eupatorium adenophorum petroleum ether extract. The acaricidal activity of each compound was tested against Psoroptes cuniculi in vitro. Three compounds had strong acaricidal activity against P. cuniculi in vitro. The insecticidal effect of 0.5% compound 9ß-hydroxy-ageraphorone was better than the insecticidal effect of fenvalerate, and compounds 9-oxo-ageraphorone and 9-oxo-10,11-dehydro-ageraphorone exhibited higher insecticidal effects than 9ß-hydroxy-ageraphorone. Thus, the E. adenophorum petroleum ether extract contains an effective composition of acaricides that could potentially be developed as a promising plant-origin acaricide.

Acaricidal activity of petroleum ether extracts from Eupatorium adenophorum against the ectoparasitic cattle mite, Chorioptes texanus.

In this study, we evaluated the acaricidal efficacy of extracts obtained from the plant Eupatorium adenophorum against the common cattle mite Chorioptes texanus. The results showed that 95% ethanol extracts at concentrations of 1.0, 0.5, and 0.25 g/mL (w/v) were highly toxic to C. texanus in vitro, killing 100% of mites in 4 h. Similarly, petroleum ether extracts of E. adenophorum resulted in between 80 and 100% mortality of mites in vitro at concentrations of 0.1, 0.05, and 0.025 mL/mL (v/v) within 4 h. In clinical trials, all infected individuals completely recovered after two treatments administered at 7-day intervals and remained disease-free at 60 days posttreatment. The clinical effect of treatment with E. adenophorum petroleum ether extracts was similar to that of treatment with the acaricide fenvalerate. These results indicated that E. adenophorum contains novel potential acaricidal compounds that can effectively control mites in livestock.

Evaluation acaricidal efficacy of botanical extract from Eupatorium adenophorum against the hard tick Haemaphysalis longicornis (Acari: Ixodidae).

The aim of this study was to evaluate the acaricidal activity of a botanical extract from Eupatorium adenophorum against the hard tick Haemaphysalis longicornis. This could result in developing effective extracts of E. adenophorum as a source of natural, low-toxicity plant-based acaricidal drugs. Adult engorged females of H. longicornis were collected from naturally infected goats. The engorged females were reared in the laboratory and their offspring (larvae and nymphs) were used as test ectoparasites. The toxic effects of botanical extracts from E. adenophorum against larvae and nymphs of H. longicornis were evaluated. The results showed that the extracts with 1.5 and 1.0g/ml (w/v) concentrations were toxic for H. longicornis, comparable to a toxic effect of 2% chlorpyrifos (positive control). The median lethal time (LT50) for larval and nymphal ticks with 1.5g/ml (w/v) concentration of extract were 0.790 (LT99=1.065) and 1.018 (LT99=10.608) hours, respectively, whereas the LT50 of 1.0g/ml (w/v) concentration were 1.445 (LT99=6.047) and 1.313 (LT99=29.932) hours for larval and nymphal ticks, respectively. At a concentration of 1.5g/ml (w/v), an acaricidal effect of 100% was achieved for both larval and nymphal ticks, while a concentration of 1.0g/ml (w/v) resulted in 100% (for larvae) and 93% (for nymphs) within a 6h period. In additional, we found that the relatively low concentration (0.5g/ml) also obtained a good acaricidal effect during the short experimental period, with 2.22 and 2.651h LT50 for larval and nymphal ticks, respectively. These results indicate that E. adenophorum contains potent acaricidal ingredients against the hard tick H. longicornis.

Analysis of the genetic diversity of the nematode parasite Baylisascaris schroederi from wild giant pandas in different mountain ranges in China.

BACKGROUND: Baylisascaris schroederi is one of the most common nematodes of the giant panda, and can cause severe baylisascarosis in both wild and captive giant pandas. Previous studies of the giant pandas indicated that this population is genetically distinct, implying the presence of a new subspecies. Based on the co-evolution between the parasite and the host, the aim of this study was to investigate the genetic differentiation in the B. schroederi population collected from giant pandas inhabiting different mountain ranges, and further to identify whether the evolution of this parasite correlates with the evolution of giant pandas. METHODS: In this study, 48 B. schroederi were collected from 28 wild giant pandas inhabiting the Qinling, Minshan and Qionglai mountain ranges in China. The complete sequence of the mitochondrial cytochrome b (mtCytb) gene was amplified by PCR, and the corresponding population genetic diversity of the three mountain populations was determined. In addition, we discussed the evolutionary relationship between B. schroederi and its host giant panda. RESULTS: For the DNA dataset, insignificant Fst values and a significant, high level of gene flow were detected among the three mountain populations of B. schroederi, and high genetic variation within populations and a low genetic distance were observed. Both phylogenetic analyses and network mapping of the 16 haplotypes revealed a dispersed pattern and an absence of branches strictly corresponding to the three mountain range sampling sites. Neutrality tests and mismatch analysis indicated that B. schroederi experienced a population expansion in the past. CONCLUSIONS: Taken together, the dispersed haplotype map, extremely high gene flow among the three populations of B. schroederi, low genetic structure and rapid evolutionary rate suggest that the B. schroederi populations did not follow a pattern of isolation by distance, indicating the existence of physical connections before these populations became geographically separated.

A sensitive and specific PCR assay for the detection of Baylisascaris schroederi eggs in giant panda feces.

Baylisascaris schroederi is one of the most common intestinal nematodes in giant pandas. It can cause severe baylisascariasis which is highly infectious in its natural hosts. A rapid and reliable diagnosis of parasite infections is crucial to protect giant pandas, as well as for environmental monitoring and disease surveillance. Here, we established a specific PCR assay for B. schroederi detection which was targeting a 331-bp long fragment of the mitochondrial cytochrome c oxidase subunit II (COII) gene. Fifty fresh fecal samples collected from captive giant pandas were tested by the established PCR assay and the traditional flotation technique. DNA extracted from a single B. schroederi egg could be successfully amplified, while no cross-reactivity was found with DNA from Ancylostoma caninum eggs. The detection rate of the PCR assay was 68%, which was higher than that of the traditional egg flotation (46%). Our findings demonstrated that the PCR assay is sensitive and specific for the detection and identification of B. schroederi eggs. Therefore, it could become a useful tool for the investigation of B. schroederi infections in giant pandas.

Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences.

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.

Cloning and characterization of the fatty acid-binding protein gene from the protoscolex of Taenia multiceps.

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.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the Sarcoptes scabiei (Sarcoptidae: Sarcoptes) in rabbits.

The aims of present study were to evaluate the therapeutic efficacy of extracts from Eupatorium adenophorum against Sarcoptes scabiei. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with S. scabiei in the toes (n=30) or artificially infected in the external ear margin with S. scabiei (n=30). Rabbits were randomly divided into five groups (6 animals per group, A-E groups for rabbits of naturally infested and F-J groups for artificially infected rabbits), respectively. All 60 rabbits were treated twice on days 0 and 7 successively. Animals in groups A/F, B/G, and C/H were treated on each toe/external ear margin with topical E. adenophorum ethanol extract at 1.0, 0.5 and 0.25 g/ml (w/v), respectively. Animals in groups D/I and E/J were treated with ivermectin by injections (positive controls) or by glycerol with water only rubbed onto the affected area (negative controls). After two treatments with extracts of E. adenophorum with relatively high concentrations of 0.5 and 1g/ml, the S. scabiei was completely eliminated in rabbits between days 14 and 30. Our results showed that rabbits treated with ivermectin (positive controls) and those treated with the extracts of concentrations of 1.0 or 0.5 g/ml achieved remarkable therapeutic efficacy; no mites were present in toes of rabbits in these groups on day 14, which confirmed a 100% therapeutic efficacy rate up to day 30 of the end of the trial. The clinical effects of treatment with 1.0 and 0.5 g/ml E. adenophorum extracts (groups A and B) were similar to ivermectin treatment. However, the therapeutic efficacy in group C and E rabbits only reached 43.25% and 7.13% by day 14. Furthermore, the therapeutic efficacy improved slightly by the end of the experiment on day 30, and rabbits in groups F, G and I also achieved good efficacy according to the recovery scoring criteria. These results indicate that E. adenophorum contains potent compounds for the effective control of sarcoptidosis.

Molecular diagnosis of Baylisascaris schroederi infections in giant panda (Ailuropoda melanoleuca) feces using PCR.

The helminth Baylisascaris schroederi is one of the most harmful parasites infecting giant pandas (Ailuropoda melanoleuca). It is therefore important to develop an exact diagnostic technique to detect this parasite. Using a known number (1, 2, 3, 4, 5, 10, 25, 50, 100) of feces-isolated B. schroederi egg and adult DNA, we developed a PCR to detect a portion of the mitochondrial 12S rRNA and applied it to giant panda fecal samples. The method was sufficiently sensitive to detect B. schroederi DNA from isolated eggs in a fecal sample with a detection threshold of one egg. We detected B. schroederi in 88% of fecal samples, 30% higher than the conventional flotation technique. No cross-reactivity with other common nematode DNA was detected. Our PCR assay may constitute a valuable alternative for the diagnosis of B. schroederi infections.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the scab mite, Psoroptes cuniculi.

This study evaluated the in vivo clinical efficacy of Crofton weed (Eupatorium adenophorum) extracts against the scab mite, Psoroptes cuniculi. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups (6 animals per group); animals in groups A, B and C were treated in each ear topically with 2 ml of 1.0, 0.5 and 0.25 g/ml (w/v) E. adenophorum ethanol extract, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7, 14 and 30, to inspect the presence or absence of mites and scabs/crusts. Clinical infection and the degree of recovery were evaluated, and the rate of reduction in mites and clinical efficacy rate (%) were calculated. The clinical effect of treatment with E. adenophorum extracts was similar to treatment with ivermectin. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.32, 3.08 and 3.17 to 0.37, 0.47 and 0.48 in the left ears of animals in groups A, B and C, respectively, and from 3.53, 3.73 and 3.67 to 0.40, 0.45 and 0.48 in the right ears of animals in groups A, B and C, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not decrease significantly (P>0.05) during the experiment, and this changed from 3.32 to 2.75 in the left ears and from 3.50 to 3.25 in the right ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (7 days space), the rabbits in groups A, B, C and D had recovered completely 30 days after the last treatment and no recurrences of infection were observed. These results indicate that E. adenophorum contains potent compounds for the effective control of animal acariasis.

Mebendazole in the treatment of Hymenolepis nana infections in the captive ring-tailed lemur (Lemur catta), China.

The present study was conducted to evaluate the effectiveness of mebendazole in the treatment of Hymenolepis nana infection in ring-tailed lemurs (Lemur catta). Ten (L. catta) from the Chengdu Zoological Garden in China, which were naturally infected with H. nana, were treated with mebendazole (10 mg/kg for 5 days). A posttreatment fecal examination was conducted 10 and 20 days after the start of treatment. All treatments resulted in a decrease in the number of eggs per gram in the posttreatment sample compared with the pretreatment sample. Reduction of mean egg count was 97.6% and 100% on days 10 and 20, respectively. The results indicated that mebendazole has marked efficacy against H. nana infections in L. catta.