Application of molecular tools in detecting zoonotic pathogens in organic fertilizers and liquid supplements.

The use of organic fertilizers and liquid supplements for crop production is rapidly growing as an alternative system to conventional agriculture. However, very little is known about the public health issues related to pathogens. This study endeavors to identify the important zoonotic pathogens with the current molecular diagnostic tools, Loop-mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA), against the conventional pathogen detection. These cost-effective molecular techniques have proven to be confirmatory tests of the target pathogens present in organic fertilizers and liquid supplements, which recommends an advancement for the comprehensive field surveillance-response approach in many developing countries with resource-limited settings quality assurance and safety implementation of organic biosolids for sustainable agricultural farming.

Evaluation of LAMP for detection and/or screening of Leptospira spp. infection among domestic animals in the Philippines.

OBJECTIVE: This study assessed the applicability of loop-mediated isothermal amplification (LAMP) for the detection of leptospirosis among domesticated animals and sewage rats. Specifically, it evaluated the ability of LAMP to amplify Leptospira spp. targeting the 16s rRNA gene in boiled urine samples. MATERIALS AND METHODS: A total of 140 samples from different domestic animals were tested for the presence of the antigen. A nested-polymerase chain reaction (nPCR) protocol was used to compare and determine the sensitivity of LAMP in detecting Leptospira spp. The LAMP was also evaluated by comparing its amplification result using agarose gel electrophoresis and color change using dye. RESULTS: Positivity rate of Leptospira spp. antigen was 29.0% (40/140) for LAMP and 9.3% (13/140) for nPCR. Also, LAMP results for gel electrophoresis and dye color change varied in some samples that may be due to the interpretation of the result in dye color change. CONCLUSION: Overall, LAMP is a rapid, sensitive, and cost-effective diagnostic method compared with nPCR. Also, LAMP has a potential application as pen-side screening, surveillance, and clinical diagnostic kits of infectious diseases without requiring advance equipment and skilled personnel.

Detection of Cryptosporidium parvum DNA in fecal samples of infected cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in the Philippines using loop mediated isothermal amplification method

Fecal DNA samples from 17 cattle and 38 water buffaloes found to be infected with Cryptosporidium oocysts using Kinyoun acid fast stain from a previous study, were subjected to loop-mediated isothermal amplification (LAMP) assay using specific primers for Cryptosporidium parvum (C. parvum) from three municipalities, Maria, Baler and San Luis of the province of Aurora in the Philippines. Results of the fecalysis using Kinyoun acid fast stain and LAMP assay were compared with the PCR results of the examined farmer/owner who raised these animals to determine the possible zoonoses of C. parvum between the farmers and their animals. Using LAMP assay, only 41% (7/17) were positive in cattle and 76% (29/38) in water buffaloes. Out of the seven LAMP positive cases in cattle, 86% (6/7) came from Maria and 14% (1/7) from Baler. Out of 29 LAMP positive cases in water buffaloes, 62% (18/29) came from Maria, 24% (7/29) from Baler and 14% (4/29) from San Luis. Comparing with the earlier results for probable zoonoses of C. parvum between the farmers and their animal was determined. Eight farmers that were positive in PCR and with their water buffaloes, positive in LAMP assay were detected to have C. parvum. Only one farmer with his cattle was detected positive of Cryptosporidium spp. in PCR, however, it was negative in LAMP assay hence, a non-parvum species might infected the farmer and the animal.

Phylogenetic tracking of current porcine epidemic diarrhea virus (PEDV) strains in the Philippines.

To trace the possible route of introduction of porcine epidemic diarrhea virus (PEDV), the phylogenetic relationships of PEDV strains in the regions of epidemicity in the Philippines to PEDV strains that are endemic in other countries were investigated. Partial nucleotide sequences of the S1 spike gene was determined from the PEDV-positive samples and compared with S1 sequences from other countries. Phylogenetic analysis indicated that PEDV strains in the Philippines segregate into two groups. Members of group 1 are related to strains from the USA, Taiwan, Japan and Canada, while those in group 2 are related to strains from China and Vietnam.