Schistosoma japonicum: A PCR assay for the early detection and evaluation of treatment in a rabbit model

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8 pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

DNA Detection of Schistosoma japonicum: Diagnostic Validity of a LAMP Assay for Low-Intensity Infection and Effects of Chemotherapy in Humans

Background

Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP) for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum.

Methodology/ Principal Findings

LAMP was first assessed in rabbits with low intensity infection (EPG<10). Then 110 patient sera from Hunan Province, China, and 47 sera after treatment by praziquantel were used to evaluate the diagnostic validity of LAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy” residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3^rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 “healthy” individuals from endemic area, 10 (16.7%) individuals were positive by LAMP, which suggested that these individuals might be false-negative patients.

Conclusions/ Significance

The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted treatment of individuals, particularly for those with negative stool examinations who may yet remain infected. The LAMP assay may provide a potential tool to support schistosomiasis control and elimination strategies.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP)

Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10^− 3 ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.     

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 min at 63 °C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T_m) of ∼89 °C. The assay analytical sensitivity is ∼0.1 tryps/ml while that of classical PCR test targeting the same gene is ∼10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.     

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/μL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.     

A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

Background

Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.

Methodology/Principal findings

A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.

Conclusions/Significance

We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.     

Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis

Background

Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens.

Methodology/Principal Findings

In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients.

Conclusions/Significance

Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful molecular diagnostic techniques that can be used for monitoring early infection and therapy efficacy to support schistosomiasis control programs.     

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.     

Quantitative Detection of Schistosoma japonicum Cercariae in Water by Real-Time PCR

In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R² = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas.     

Functional properties of hemocyanin from Oncomelania hupensis, the intermediate host of Schistosoma japonicum

The gastropod mollusc, Oncomelania hupensis is a unique intermediate host for the human parasite Schistosoma japonicum. It is a primary factor for the epidemic of schistosomiasis and its distribution is consistent with the epidemic area of schistosomiasis. Here we report the functional properties of hemocyanin of O. hupensis (OhH), a copper-containing respiratory protein which was isolated from its hemolymph and purified by ammonium sulfate fractionation and ultracentrifugation. We identified the protein characters including UV absorption at 340 nm, copper content and quaternary structure. Furthermore, by induction of phenoloxidase and enzyme-linked immunosorbent assay we show that OhH exhibited o-diphenoloxidase activity after limited proteolysis, and shared carbohydrate epitopes with glycoconjugates of S. japonicum.     

Geographical distribution of human Schistosoma japonicum infection in The Philippines: tools to support disease control and further elimination

Schistosoma japonicum infection is believed to be endemic in 28 of the 80 provinces of The Philippines and the most recent data on schistosomiasis prevalence have shown considerable variability between provinces. In order to increase the efficient allocation of parasitic disease control resources in the country, we aimed to describe the small-scale spatial variation in S. japonicum prevalence across The Philippines, quantify the role of the physical environment in driving the spatial variation of S. japonicum, and develop a predictive risk map of S. japonicum infection. Data on S. japonicum infection from 35,754 individuals across the country were geo-located at the barangay level and included in the analysis. The analysis was then stratified geographically for the regions of Luzon, the Visayas and Mindanao. Zero-inflated binomial Bayesian geostatistical models of S. japonicum prevalence were developed and diagnostic uncertainty was incorporated. Results of the analysis show that in the three regions, males and individuals aged ?20 years had significantly higher prevalence of S. japonicum compared with females and children <5 years. The role of the environmental variables differed between regions of The Philippines. Schistosoma japonicum infection was widespread in the Visayas whereas it was much more focal in Luzon and Mindanao. This analysis revealed significant spatial variation in the prevalence of S. japonicum infection in The Philippines. This suggests that a spatially targeted approach to schistosomiasis interventions, including mass drug administration, is warranted. When financially possible, additional schistosomiasis surveys should be prioritised for areas identified to be at high risk but which were under-represented in our dataset.     

Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay (‘GenoType MTBC’ from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay (‘MTB LAMP’) showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay (‘Muniv LAMP’) showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.     

The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China

The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 μl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.     

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.     

Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.     

Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.