Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

QCM-based rapid detection of PCR amplification products of Ehrlichia canis.

Ehrlichia canis is an intracellular parasitic bacterium and arthropod-borne pathogen that receives growing attention, because it leads to increasing morbidity and mortality in animals. It does so by causing canine monocytotropic ehrlichiosis (CME). Infected canines may lack obvious clinical signs and stay in chronic stage. Herein we report a rapid screening method based on PCR assay combined with quartz crystal microbalance (QCM) to design a DNA sensor for detecting E. canis in early stages of infection. The test relies on DNA amplification of target nucleotide sequences via PCR followed by detecting DNA-DNA hybridization using QCM. The approach did not result in any cross-hybridization toward other blood bacteria or parasites in dogs, such as Anaplasma platys, Babesia canis and Trypanosoma spp, but turned out selective for the target species. The limit of detection of QCM was as low as 4.1 × 109 molecules/µl of 289 bp E. canis PCR products corresponding to 22 copy numbers/µl of E. canis. Furthermore, the technique is also simple, does not require complicated equipment and can in principle be reused.

Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells.

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 µg/ml) for 6-48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18-24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

Third-stage Gnathostoma spinigerum larva excretory secretory antigens modulate function of Fc gamma receptor I-mediated monocytes in peripheral blood mononuclear cell culture.

BACKGROUND: Third (infective)-stage Gnathostoma spinigerum larvae (L3) mainly cause human gnathostomiasis. G. spinigerum L3 migrate throughout the subcutaneous tissues, vital organs, and central nervous system and can cause various pathogenesis including sudden death. Interestingly, G. spinigerum L3 can survive and evade host cellular immunity for months or years. The effects of G. spinigerum excretory-secretory (ES) products involved in larval migration and immune-evasive strategies are unknown. Monocytes are innate immune cells that act as phagocytic and antigen-presenting cells and also play roles against helminthic infections via a complex interplay between other immune cells. Fc gamma receptor I (FcγRI) is a high-affinity receptor that is particularly expressed on monocytes, macrophages, and dendritic cells. The cross-linking of FcγRI and antigen-antibody complex initiates signal transduction cascades in phagocytosis, cytokine production, and antibody-dependent cell-mediated cytotoxicity (ADCC). This study investigated whether ES antigen (ESA) from G. spinigerum L3 affects monocyte functions. RESULTS: Cultures of normal peripheral blood mononuclear cells (PBMC) separated from healthy buffy coats were used as a human immune cell model. ESA was prepared from G. spinigerum L3 culture. Using Real-Time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the effect of ESA to down-regulate FcγRI mRNA expression in monocytes during 90 min of observation was not well delineated. Flow cytometry analysis revealed a significant phenotypic-decreased FcγRI expression on the monocyte surface at 12 hours (h) of cultivation with the ESA (p = 0.033). Significantly reduced monocyte-mediated phagocytosis capacity was consistently observed after 12 h of ESA pretreatment (p = 0.001). CONCLUSIONS: Our results suggest that G. spinigerum ESA modulates monocyte function via depletion of FcγRI expression. This study provides preliminary information for future in-depth studies to elucidate mechanisms of the immune-evasive strategy of G. spinigerum larvae.

Comparative Studies on Nucleic Acid Based Biosensors for Identification of Filarial Nematode.

Background: The microfilarial nematodes, found mainly in blood circulation, form a special group of human and veterinary parasitic diseases. Recently, the rapid nucleic acid based biosensors (NABs) have been established in our laboratory for the employment of a point-of-care diagnostic test. Herein, NABs involved in the exploitation of lateral flow dipstick (LFD), gold nanoparticle (AuNP) and turbidity in combination with PCR and LAMP amplification for detection of filarial nematodes. The validation of each NABs was investigated in comparison to that of standard detection methods using the same unknown blood specimens. Objective: To compare the sensitivity, specificity and accuracy of NABs with standard detection methods. Material and Method: In this study, the microfilariae of Dirofilaria immitis was used as the representative model for filarial nematode. The PCR and LAMP primers were designed and synthesized according to the specific nucleotide regions of a small subunit gene of the parasite as well as the DNA probes. The fifty unknown blood samples were sent as a gift from Prasu Arthorn Animal Hospital, Faculty of Veterinary Science, Mahidol University. The samples were tested by using PCR, PCRLFD, PCR-AuNP, LAMP, LAMP-LFD, LAMP-AuNP, LAMP-turbidity and film blood smear. The efficacy of NABs was compared to a standard film blood smear in terms of sensitivity, specificity and accuracy. Results: Upon detection of fifty unknown blood samples, LAMP-LFD assay presented 100% of sensitivity, specificity and accuracy. The data revealed that sensitivity, specificity and accuracy of NABs varied from 66.67-100.00% when compared to a film blood smear stained with Giemsa dye. The data clearly indicated that LAMP-LFD was a preferred choice for the use as a point-of-care NABs. Conclusion: NABs were highly sensitive performing as a selective diagnostic tool that could be applied particularly as the rapid screening tests for filarial nematodes hence as a recommended epidemiological survey.

Isolation and characterization of microsatellite markers from the great hornbill, Buceros bicornis.

Thirteen polymorphic microsatellite markers were isolated and characterized from the great hornbill, Buceros bicornis. In analyses of 20 individuals, the numbers of alleles per locus varied from two to 11. The expected and observed heterozygosities ranged from 0.22 to 0.88 and from 0.20 to 1.00, respectively. The mean polymorphic information content was 0.62. Among these, three loci deviated from the Hardy-Weinberg equilibrium. However, no significant genotypic disequilibrium was detected between any pair of loci. These microsatellite markers are useful for the population genetic study of the great hornbill.

Genetic diversity of Trypanosoma evansi in buffalo based on internal transcribed spacer (ITS) regions.

The nucleotide sequences of 18S rDNA and internal transcribed spacer (ITS) regions were used for studying the relationships of Trypanosoma evansi isolate from a buffalo. The sequences were analyzed and compared to 18S rDNA and the ITS regions of the other Trypanosoma spp. Maximum likelihood phylogenetic trees were constructed using Leishmania major as the outgroup. The tree of 18S rDNA indicated that T. evansi (buffalo B18) isolate was closely related to those of Taiwan and T. brucei stock. The ITS tree showed the genetic diversity among 32 clones of T. evansi (B18) within a single host. This data will be useful for epidemiological and dynamic studies for designing the rational control programs of the disease.

The observation of microfilarial rate and density in cats inoculated with increasing numbers of Brugia pahangi infective larvae.

Having close kinship to Brugia malayi, B. pahangi is a member of the family Filariidae, which causes lymphatic filariasis in dogs and cats. Although this nematode is unlikely to cause a zoonotic disease in humans, study of the B. pahangi life cycle may help control human filariasis. The objective of this study was to examine microfilarial rates and densities of B. pahangi in experimentally induced infections in cats as a relative measurement. Cats were infected with 3 different amounts of 3rd-stage larvae (L3); 100, 300 and 500. Cats infected with 100 L3 became patent for microfilariae longer than the other groups (mean100 = 99+/-44 days). In comparison, the pre-patent period of B. pahangi was somewhat shorter in cats with 300 and 500 L3 infections (mean300 = 76+/-13 and mean500 = 63+/-5 days). The microfilarial densities of these cats were also determined; the density of microfilariae (mf/1 ml blood) increased relative to the duration of infection. One-way ANOVA tests were used to compare the microfilarial densities of the cats with varying numbers of L3. We found that the microfilarial density of cats with 500 L3 exhibited significant differences (p < 0.05) from cats with 300 and 100 L3. However, we concluded that the amount of microfilariae produced in the blood circulation of these cats were not increasing relative to the numbers of L3 taken by the host.

The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand.

In this report, the genome of the Thai avian influenza virus A (H5N1); A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04, isolated from the Thai avian influenza A (AI) epidemic during the early of 2004 was sequenced. Phylogenetic analyses were performed in comparison to AI viruses from Hong Kong 1997 outbreaks and other AI (H5N1) isolates reported during 2001-2004. Molecular characterization of the Thai AI (H5N1) HA gene revealed a common characteristic of a highly pathogenic AI (HPAI), a 20-codon deletion in the neuraminidase gene, a 5-codon deletion in the NS gene and polymorphisms of the M2 and PB2 genes. Moreover, the HA and NA genes of the Thai AI displayed high similarity to those of the AI viruses isolated from human cases during the same epidemic. Finally, our results demonstrated that the Thai AI emerged as a member of 2000's AI lineage with most of the genetic sequences closely related to the Influenza A/Duck/China/E319.2/03 (H5N1).