Transcriptome profiling of Cysticercus Pisiformis provides insight into responses to host bile acids.

Bile acids in host intestine activate larvae of tapeworms and facilitate its invasion. However, the mechanism underlying this process is poorly understood. In order to better understand responses of tapeworms to host biles, we used RNA-Seq profiling method to study the transcriptomes of Cysticercus Pisiformis (larvae of Taenia Pisiformis) after host bile acid treatment. A total of 338.32 million high-quality clean reads were obtained by Illumina Hiseq platform. Totally, 62,009 unigenes were assembled, 38,382 of which were successfully annotated to known databases. A total of 9324 unigenes were identified as differentially expressed genes (DEGs), of which 5380 and 3944 genes were up- and down-regulated in the group treated with bile acids, respectively. Gene Ontology analysis revealed that biosynthesis and energy metabolism potential were significantly strengthened after host bile treatment in C. pisiformis. Similarly, KEGG pathway analysis revealed an enrichment of pathways related to lipid metabolism and carbohydrate metabolism. Among them, 'AMPK signaling pathway' which is critical in balancing cellular energy, was significantly enriched after bile acids activation. In addition, pathways of 'Fatty acid biosynthesis', 'Fatty acid elongation', 'Starch and sucrose metabolism', and 'glycolysis gluconeogenesis' were also significantly changed after bile acid treatment. qRT-PCR analysis confirmed the differential abundances of some key genes in these pathways. Our data suggest that host bile acids remarkably promote the pathways of energy metabolism of this parasite and regulate the genes involved in balancing lipid metabolism and carbohydrate metabolism. These findings provide new insights on the lifecycle of Taenia parasites.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus.

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, ß-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (ß-strand â†’ coil, coil â†’ ß-strand, α-helix â†’ coil, coil â†’ α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (ß-strand â†’ coil and coil â†’ ß-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.

The Regulatory Roles of ncRNA Rli60 in Adaptability of Listeria monocytogenes to Environmental Stress and Biofilm Formation.

Listeria monocytogenes is a facultative anaerobic Gram-positive bacterium. It is well adapted to external environments and able to infect both humans and animals. To understand the impacts of ncRNA Rli60 on the adaptability of L. monocytogenes to environmental stresses and biofilm formation, a rli60 deletion strain of L. monocytogenes (LM-Δrli60) was constructed using splicing by overlap extension PCR (SOE-PCR) and homologous recombination and then compared it with wild-type strain L. monocytogenes EGD-e in the aspects of adaptability to environmental stresses by measuring their growth under stresses of different temperatures, and acidic, alkaline, hypertonic and alcoholic conditions, and capability of biofilm formation by using crystal violet staining, as well as the transcriptional levels of genes (gltB and gltC) related to the biofilm formation by real-time quantitative PCR (qRT-PCR). The results showed that (1) the growth of LM-Δrli60 strain was significantly slower under environmental stresses of low temperature (30 °C), high temperature (42 °C), as well as alkaline and alcoholic conditions, (2) the amount of biofilm formed by LM-Δrli60 was attenuated, and (3) the transcriptional levels of gltB and gltC genes at 24 h and 48 h in LM-Δrli60 revealed a significant reduction. Overall, the results confirmed that ncRNA Rli60 plays important roles in regulating the adaptability of L. monocytogenes to environmental stresses and biofilm formation possibly through impacting the expression of gltB and gltC genes.

The roles of noncoding RNA Rli60 in regulating the virulence of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes (LM) is an important zoonotic foodborne pathogen. Noncoding RNA (ncRNA) has an important role in regulating its virulence. As a member of ncRNA, however, the function of Rli60 in regulating LM virulence remain unclear. The aim of this study was to investigate the role of Rli60 in regulating LM virulence. METHODS: Using a homologous recombination method, a LM EGD-e rli60 gene deletion strain (LM-Δrli60) was constructed and compared with a LM EGD-e strain in the following respects: (1) adhesiveness, invasion ability, intracellular survival, proliferation, and transcription of virulence genes in the mouse macrophage cell line RAW264.7; (2) 50% lethal dose (LD50) to the BALB/c mouse; and (3) the amount in the mouse liver and spleen and the effects on pathology of mouse liver, spleen, and kidney after inoculation. RESULTS: The LM-Δrli60 strain had a significantly higher adhesion rate and lower invasion rate with significantly lower intracellular survival and proliferation rates in the RAW264.7 cell line, compared to the LM EGD-e strain. Inoculation with LM-Δrli60 strain significantly affected the transcription of virulence genes. The LD50 of LM-Δrli60 to BALB/c mouse was increased by 2.12 logarithmic magnitude, which indicated that the virulence in LM-Δrli60 is significantly decreased (p < 0.05). The amount of LM-Δrli60 in the liver and spleen was significantly lower than the amount of LM EGD-e in these organs (p < 0.05). The pathological damage due to LM-Δrli60 infection in the mouse liver, spleen, and kidney was lower than the damage due to LM EGD-e infection. CONCLUSION: This study confirmed that the rli60 deletion could significantly affect LM virulence, adhesion, invasion, survival, and proliferation. This suggests that Rli60 has an important role in regulating LM virulence.

[Cloning of Taenia pisiformis Actin Gene and Assessment of Its Use as An Internal Control].

Objective: To clone the full-length cDNA of actin gene of Taenia pisiformis (Tp-actin), and analyze the gene structure, phylogenetic evolution and its use as an internal control. Methods: Tp-actin was amplified by RT-PCR and the cDNA of 3' and 5' ends were obtained through RACE-PCR. After sequencing, these segments were linked to produce full-length cDNA of Tp-actin. The gene structure and phylogenetic evolution were analyzed using bioinformatics software. Primers for Tp-actin and cysteine peptidase (TpCP) were designed using Primer Express software. Primer specificity and amplification efficiency were analyzed with real-time fluorescence quantitative PCR (qRT-PCR). In addition, by using Tp-actin as an internal control, the expression of TpCP in T. pisiformis at various developmental stages was analyzed. Results: As expected, sequencing results showed that the Tp-actin fragment was 1 048 bp in length, and the 3' and 5' ends were 428 bp and 945 bp, respectively. The full-length cDNA of Tp-actin generated from the 3 segments (submitted to GenBank with accession No. JX624787) was 1 279 bp, containing a 30-bp 5'-untranslated region（5'-UTR）, a 118-bp 3'-UTR, and a 1 131-bp open reading frame (ORF). Bioinformatics analysis showed that the Tp-actin encoded a protein of 356 amino acids, with a predicted relative molecular weight of 41 749 and a PI value of 5.29. This protein was predicted to contain 6 functional sites and 3 typical signatures of the actin family. Phylogenetic analysis showed that the Tp-actin was 100% and 99.7% homologous in amino acid sequence to those of Taenia solium and Diphyllobothrium dendriticum. qRT-PCR resulted in specific products of 82 bp and 108 bp from Tp-actin and TpCP, respectively, melting curves of which both showed a single signal peak, verifying the high specificity of primers. The linear correlation coefficient（R2） in standard curve of Tp-actin was 0.999, showing high amplification efficiency. Using Tp-actin as the internal control, the relative expression ratio of TpCP gene in gravid proglottid of T. pisiformis（1.65） was significantly higher than that in oncospheres （1.00）, mature proglottids （0.87） and cysticercus (0.62) (P<0.05). Conclusion: Tp-actin gene is highly conserved and can be used as a reliable internal control.

Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain.

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is a highly contagious chicken disease, and can lead to serious economic losses in poultry enterprises. The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants. Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines. In this study, the recombinant IBV is developed by replacing the ectodomain region of the S1 gene of the IBV Beaudette strain with the corresponding fragment from H120 strain, designated as rBeau-H120(S1e). In Vero cells, the virus proliferates as its parental virus and can cause syncytium formation. The peak titer would reach 10(5.9) 50% (median) tissue culture infective dose/mL at 24 h post-infection. After inoculation of chickens with the recombinant virus, it demonstrated that rBeau-H120(S1e) remained nonpathogenic and was restricted in its replication in vivo. Protection studies showed that vaccination with rBeau-H120 (S1e) at 7-day after hatch provided 80% rate of immune protection against challenge with 10(3) 50% embryos infection dose of the virulent IBV M41 strain. These results indicate that rBeau-H120 (S1e) has the potential to be an alternative vaccine against IBV based on excellent propagation property and immunogenicity. This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.

The mitochondrial genome of Paramphistomum cervi (Digenea), the first representative for the family Paramphistomidae.

We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.

Mapping codon usage of the translation initiation region in porcine reproductive and respiratory syndrome virus genome.

BACKGROUND: Porcine reproductive and respitatory syndrome virus (PRRSV) is a recently emerged pathogen and severely affects swine populations worldwide. The replication of PRRSV is tightly controlled by viral gene expression and the codon usage of translation initiation region within each gene could potentially regulate the translation rate. Therefore, a better understanding of the codon usage pattern of the initiation translation region would shed light on the regulation of PRRSV gene expression. RESULTS: In this study, the codon usage in the translation initiation region and in the whole coding sequence was compared in PRRSV ORF1a and ORFs2-7. To investigate the potential role of codon usage in affecting the translation initiation rate, we established a codon usage model for PRRSV translation initiation region. We observed that some non-preferential codons are preferentially used in the translation initiation region in particular ORFs. Although some positions vary with codons, they intend to use codons with negative CUB. Furthermore, our model of codon usage showed that the conserved pattern of CUB is not directly consensus with the conserved sequence, but shaped under the translation selection. CONCLUSIONS: The non-variation pattern with negative CUB in the PRRSV translation initiation region scanned by ribosomes is considered the rate-limiting step in the translation process.

A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A.

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7 ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatitis virus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.

Promising multiple-epitope recombinant vaccine against foot-and-mouth disease virus type O in swine.

In order to develop a completely safe immunogen to replace the traditional inactivated vaccine, a tandem-repeat multiple-epitope recombinant vaccine against foot-and-mouth disease (FMD) virus (FMDV) type O was developed. It contained three copies each of residues 141 to 160 and 200 to 213 of VP1 of the O/China/99 strain of FMDV coupled with a swine immunoglobulin G heavy-chain constant region (scIgG). The data showed that the multiple-epitope recombinant vaccine elicited high titers of anti-FMDV specific antibodies in swine at 30 days postvaccination (dpv) and conferred complete protection against a challenge with 10³ 50% swine infective doses of the O/China/99 strain. The anti-FMDV specific antibody titers were not significantly different between the multiple-epitope recombinant vaccine and the traditional vaccine (t test, P > 0.05). The number of 50% pig protective doses was 6.47, which is higher than the number recommended by the World Organization for Animal Health. The multiple-epitope recombinant vaccine resulted in a duration of immunity of at least 6 months. We speculate that the multiple-epitope recombinant vaccine is a promising vaccine that may replace the traditional inactivated vaccine for the prevention and control of FMD in swine in the future.

Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens.

We developed a rapid immunochromatographic strip (ICS) procedure that can detect circulating antigens in the blood of animals during the acute stage of toxoplasmosis. The aim of this study was to evaluate this test using sera from field samples and from experimentally infected animals. The sensitivity and specificity of the ICS were compared with those of an enzyme-linked immunosorbent assay (ELISA). Both assays detected circulating antigens in the sera of animals experimentally infected with the Gansu Jingtai strain of Toxoplasma gondii, and the agreement between the two assays was 100%. In the infected animals, circulating antigens could be detected as early as the second day post-infection (PI) and in all animals by the fourth day. In the 381 field serum samples, the positive rates of the ICS and ELISA were 5.2% and 5.8%, respectively. In addition, there was no cross-reactivity of the antigens with Neospora caninum. The results presented here suggest that the ICS is a feasible, convenient, rapid and effective method to detect infection by T. gondii. This test could be a powerful supplement to the current diagnostic methods. Taken together, the results of this study encourage further research toward the production of commercial diagnostic tests for detecting T. gondii in animals.

Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2.

BACKGROUND: The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection. RESULTS: The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%. CONCLUSIONS: This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera.

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs.

Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.

[Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level.

Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance.

BACKGROUND: Mitochondrial genomes provide a rich source of molecular variation of proven and widespread utility in molecular ecology, population genetics and evolutionary biology. The tapeworm genus Taenia includes a diversity of tapeworm parasites of significant human and veterinary importance. Here we add complete sequences of the mt genomes of T. multiceps, T. hydatigena and T. pisiformis, to a data set of 4 published mtDNAs in the same genus. Seven complete mt genomes of Taenia species are used to compare and contrast variation within and between genomes in the genus, to estimate a phylogeny for the genus, and to develop novel molecular markers as part of an extended mitochondrial toolkit. RESULTS: The complete circular mtDNAs of T. multiceps, T. hydatigena and T. pisiformis were 13,693, 13,492 and 13,387 bp in size respectively, comprising the usual complement of flatworm genes. Start and stop codons of protein coding genes included those found commonly amongst other platyhelminth mt genomes, but the much rarer initiation codon GTT was inferred for the gene atp6 in T. pisiformis. Phylogenetic analysis of mtDNAs offered novel estimates of the interrelationships of Taenia. Sliding window analyses showed nad6, nad5, atp6, nad3 and nad2 are amongst the most variable of genes per unit length, with the highest peaks in nucleotide diversity found in nad5. New primer pairs capable of amplifying fragments of variable DNA in nad1, rrnS and nad5 genes were designed in silico and tested as possible alternatives to existing mitochondrial markers for Taenia. CONCLUSIONS: With the availability of complete mtDNAs of 7 Taenia species, we have shown that analysis of amino acids provides a robust estimate of phylogeny for the genus that differs markedly from morphological estimates or those using partial genes; with implications for understanding the evolutionary radiation of important Taenia. Full alignment of the nucleotides of Taenia mtDNAs and sliding window analysis suggests numerous alternative gene regions are likely to capture greater nucleotide variation than those currently pursued as molecular markers. New PCR primers developed from a comparative mitogenomic analysis of Taenia species, extend the use of mitochondrial markers for molecular ecology, population genetics and diagnostics.

Simple method of monoclonal antibody production against mammalian cellular prion protein.

Monoclonal antibodies (MAbs) against prion protein (PrP) are powerful tools for diagnosis and research in transmissible spongiform encephalopathies. Ten MAbs to recombinant/native cellular PrP (PrPc) in mammals were prepared with a simple method and identified in detail. Normal BALB/c mice were immunized with the recombinant bovine mature PrP (rbomPrP) and PrP27-30 (rboPrP27-30) expressed in Escherichia coli. The immunized splenocytes were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA). The characterizations of these MAbs, such as Ig, Ig subclass, titer, affinity index, specificity, epitopes recognized, and binding to recombinant/native PrPc of cattle, sheep, or human beings, were evaluated by Western blotting and indirect or sandwich ELISA. Ten MAbs could be divided into five groups depending on the results of indirect ELISA additivity test and their reaction to E. coli-expressed truncated-PrPs. Isotyping of the MAbs revealed that they belong to IgG1, IgG2a, and IgG2b subclass. Their indirect ELISA titers were between 10(3) and 10(6). Affinity constants were between 10(9) and 10(12) M(-1). Ten MAbs specifically reacted with the rbomPrP, without binding to prion-like protein Doppel and the lysates of E. coli. These MAbs could also respond to the recombinant mature PrP (rmPrP) of sheep and human beings. Also of interest was the ability of the MAbs to bind with dimer of rmPrP and PrP extracted from the brain tissue of cattle or sheep. We conclude that anti-PrP MAbs successfully prepared with a simple method could potentially be useful in mammalian prion research.

Reverse transcription loop-mediated isothermal amplification for the rapid detection of infectious bronchitis virus in infected chicken tissues.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the nucleocapsid phosphoprotein gene of infectious bronchitis virus (IBV) was developed. The detection limits for the IBV RT-LAMP assay were 10(1) 50% egg infection dose (EID(50)) per 50 microl of titrated viruses and no cross-reaction of IBV RT-LAMP was found when tested with other viruses including Newcastle disease virus (NDV), avian reovirus (ARV), and infectious laryngotrachietis virus (ILTV) due to their mismatch with IBV RT-LAMP primers. A total of 187 clinical tissues samples (88 blood, 62 kidney and 37 lung) were evaluated and compared to conventional RT-PCR. The sensitivity of RT-LAMP and RT-PCR assays for detecting IBV RNA in clinical specimens was 99.5% and 98.4%, respectively. These findings showed that the RT-LAMP assay has potential usefulness for rapid and sensitive diagnosis in outbreak of IBV.

Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification.

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine parvovirus (PPV) DNA. The amplification could be finished in 45 min under isothermal condition at 62 degrees C by employing a set of four primers targeting VP2 gene of PPV. LAMP assay showed higher sensitivity than PCR, with a detection limit of 5 copies of PPV genomic DNA per reaction. No cross reactivity was observed from the samples of other related viruses including canine parvovirus, parvovirus B19, porcine circovirus type 1, porcine circovirus type 2 and porcine peudorabies virus. The detection rate of PPV LAMP for 125 clinical samples was 97.6% and appeared higher than that of PCR method. The result indicated the potential usefulness of the technique as a simple, rapid procedure for the detection of PPV.

[Investigation on echinococcosis in animals in Gannan Tibetan Autonomous Prefecture].

OBJECTIVE: To make an investigation on echinococcosis among animals in Gannan Tibetan Autonomous Prefecture. METHODS: 21 villages from Maqu and Luqu counties were selected for the survey in August of 2004-September of 2007. Rodents were trapped in the field. Sheep and yak livers, hearts and lungs were collected from the local slaughterhouses for pathological examination. Domestic dogs (shepherd dogs) were de-wormed by 15% arecoline to receive adult worms and stray dogs were shot for dissection. RESULTS: The prevalence of alveolar echinococcosis (AE) in Ochotona dahurica was 1.2% (1/87), and 2.3% (3/132) in Myospalax fontaniere, but no infection was found in Marmota himalayana, Ochotona tibetana and Mus musculus. 113 out of 1021 (11.1%) sheep were found infected with cystic echinococcosis (CE), and 3 (0.3%) with AE. 126 out of 634 (19.9%) yaks were infected with CE, and 2 yaks (0.3%) with AE. 17 out of 74 (23.0%) dogs were infected with Echinococcus granulosus (Eg), and 4 (5.4%) with Echinococcus multilocularis (Em). CONCLUSION: The results showed that there is a widespread endemic of Echinococcus granulosus in dogs and wild animals in Gannan Tibetan Autonomous Prefecture, with less Echinococcus multilocularis infection.