Now showing items 1-13 of 13

    • Article

      Biodegradition of monochloroacetic acid by a presumptive Pseudomonas sp. strain R1 bacterium isolated from Malaysian paddy (rice) field 

      SN Ismail, AM Taha, NH Jing, RA Wahab, AA Hamid, RV Pakingking Jr. & F Huyop - Biotechnology, 2008 - Asian Network for Scientific Information
      A bacterial strain tentatively identified as Pseudomonas sp. R1 was isolated from a paddy (rice) field that could degrade monochloroacetic acid (MCA) for concentrations ranging from 5 to 40 mM. Quantitative agreement between the amount of MCA introduced and chloride released was also found. MCA dehalogenase activity in this strain was found to be inducible. Cell-free extracts displayed dehalogenating activity with specific halogenated organic compound with no activity on dichloropropionic acid or monochloropropionic acid. The estimated Km values for MCA was 0.14 mM. The optimal pH range for MCA dehalogenase activity (between pH 6.5 and 8.0), whereas the thermal stability profile stable up to 50 °C. The results of our current study demonstrated the potential use of Pseudomonas sp. R1 as suitable biological agent for biodegradation of MCA in contaminated agricultural area.
    • Article

      Characteristics of dehalogenase from bacteria isolated from the gut of pond-reared rohu (Labeo rohita) juveniles in Myanmar 

      E Abel, RV Pakingking Jr., G Pagador, MT Wint & F Huyop - Advances in Bioscience and Biotechnology, 2012 - Scientific Research Publishing
      Unwarranted accumulation of halogenated compounds in the rivers and streams has in recent years emerged due to the widespread use agricultural pesticides. The presence of these halogenated compounds in the water does not only suppress the immune system of fish but adversely induce serious morbidity and mortality among cultured stocks. Importantly, gradual accumulation of these compounds in the system of cultured and wild freshwater fish species cultured in ponds and floating net-cages in dams and rivers, respectively, poses some risks to humans, the end users. In this study, we attempted to isolate bacteria from the gut of pond-reared rohu (Labeo rohita) in Myanmar, screened the isolated bacteria for dehalogenase gene using molecular technique and tested the ability of these bacteria to degrade halogenated compounds in vitro. The eight bacterial strains studied were identified as Enterobacter mori strain MK-121001, Enterobacter cloacae strains MK121003, MK-121004, MK121010, Ralstonia solanacearum strain 121002, Acinetobacter baumannii strain MK121007, Chromobacterium violaceum strain MK121009 and Pantoea vagans strain 121011. Only three bacterial strains (MK121002, MK121007 and MK121009) were capable of degrading 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon source up to a final substrate concentration of 20 mM. Their mean growth doubling time ranging from 6 - 23 hours with the maximum of chloride ion released of 85%. PCR amplification with oligonucleotide primers designed from group I dehalogenase revealed the presence of dehalogenase genes in all isolates suggesting dehalogenase gene in strains 121001, 121003, 121004, 121010 and 121011 were silenced. In contrast, group II dehalogenase primers did not show any PCR amplification. These results suggest that MK121002, MK121007 and MK121009 only encode a group I dehalogenase and its non-stereoselectivity is in agreement with previoulsly described group I haloacid dehalogenase. The partial gene sequences were blasted but no significant sequence identity was observed. Therefore, it suggests the 2-haloacid dehalogenase of MK121002, MK12-1007 and MK- 121009 might be a novel group I 2-haloacid dehalogenase. The results indicated a broad distribution of dehalogenation genes in many microbial genomes that harbor dehalogenase(s) due to the exposure of the microorganisms to the naturally occurring or man-made halogenated compounds in the environmental systems. So far, microorganisms capable of producing dehalogenases were mainly isolated from soil and scarcely from aquatic animals and their environments. To the authors’ knowledge, this is the first report on the isolation of dehalogenase-producing bacteria from the gut of pond-reared freshwater fish, Labeo rohita, in Myanmar.
    • Article

      Dehalogenase from Methylobacterium sp. HJ1 induced by the herbicide 2, 2-dichloropropionate (Dalapon) 

      NH Jing, AM Taha, RV Pakingking Jr., RAB Wahab & F Huyop - African Journal of Microbiology Research, 2008 - Academic Journals
      Heavy industrial activities and agricultural processes require consumption of many halogenated compounds, and release them continuously as pollutants into the environment. These xenobiotics show high toxicity and persistence and cause many problems to the society, soils and ground water. Microbial dehalogenases are involved in the biodegradation of many important chlorinated compounds. A bacterial strain identified as Methylobacterium sp. HJ1 is able to degrade the herbicide 2,2-dichloropropionic acid by removal of the halogen and subsequent metabolism of the product for energy. D,L-2-chloropropionate also supported good growth of the organism but 3-chloropropionate, monochloroacetate and dichloroacetate were not utilized. Cell-free extracts of the 2,2-dichloropropionate-grown bacteria converted 2,2-dichloropropionate into pyruvate with the release of two chloride ions for each molecule of pyruvate formed. This indicates the presence of dehalogenase activity in the cell-free extracts. Only 2,2-dichloropropionate and D,L-2-chloropropionate were inducers and substrates for the dehalogenase. Monochloroacetate and dichloroacetate did not serve as an inducer, whereas 3-chloropropionate was a non-substrate inducer.
    • Article

      Dietary onion and ginger enhance growth, hemato-immunological responses, and disease resistance in brown-marbled grouper, Epinephelus fuscoguttatus. 

      MJS Apines-Amar, EC Amar, JP Faisan Jr., RV Pakingking Jr. & S Satoh - Aquaculture, Aquarium, Conservation and Legislation, 2012 - Bioflux
      A 12-week (September to December 2009) feeding trial was conducted to evaluate theimmunostimulatory effects of different substances administered orally through the diet in the brown-marbled grouper, Epinephelus fuscoguttatus. Five experimental diets containing either onion, ginger, β–glucan, or vitamin C and a control diet (without immunostimulants) were fed to the fish weighing about 44 g for 12 weeks. Onion-fed fish showed significantly increased weight gain, hematocrit, and total Ig compared to the control group; however, leukocyte differential count and ROS production were unaffected. Ginger-fed fish likewise significantly increased total Ig, ROS production and lysozyme activity. However, it did not affect growth and hematocrit value. β-glucan significantly increased growth and total Ig but had no effect on the other parameters. Vitamin C significantly increased hematocrit, total Ig and ROS production but did not increase growth. Upon challenge with a bacterial pathogen Vibrio harveyi, mortality was significantly reduced in the onion, ginger and vitamin C-fed fish but not in the β–glucan-fed fish. This study demonstrated that onion and ginger could positively affect the innate immune responses and protect grouper against Vibrio harveyi infection.
    • Article

      Identification of Pseudomonas sp. strain S3 based on small subunit ribosomal RNA gene sequences. 

      AAA Hamid, S Hamdan, RV Pakingking Jr. & F Huyop - Biotechnology, 2010 - Asian Network for Scientific Information
      Pseudomonas sp. strain S3 was isolated from Paddy (rice) field agricultural area. This organism, which can utilize a halogenated compound of D,L-2-Chloropropionic acid as sole carbon and energy source, catalyses the hydrolytic dehalogenation of both D- and L- isomers of 2-Chloropropionic acid. Identification of Pseudomonas sp. S3 is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, the amplified 16S rRNA gene sequence of Pseudomonas sp. S3 (Accession No. FJ968758) was compared to other nine selected gene sequences from the same group of Pseudomonas sp. and/or dehalogenase producing bacteria using in silico method. Their phylogenetic relationships were then determined. The results were analysed using MEGA4 software to ascertain its evolutionary distance by reconstructing a phylogenetic tree of these organisms. The evolutionary history and bootstrap consensus tree were inferred using the Neighbour-Joining method from 500 replicates. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and were in the units of the number of base substitutions per site. Based on this analysis, Pseudomonas sp. S3 16S rRNA gene was closely related to the Pseudomonas chlororaphis with genetic distance 0.170 base substitutions per site. S3 gene was also compared among known dehalogenase producing bacteria 16S rRNA genes. Results suggested that S3 was closely related to the Pseudomonas sp. R1 with a genetic distance 0.040 base substitutions per site. From present study, evolutionary relationships of 16S rRNA gene of Pseudomonas sp. S3 were elegantly illustrated by phylograms, comparable to a pedigree showing which microorganisms are most closely related.
    • Article

      Immune responses of Asian sea bass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine 

      RV Pakingking Jr., R Seron, LD de la Peña, K Mori, H Yamashita & T Nakai - Journal of Fish Diseases, 2009 - Blackwell Publishing
      Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.
    • Article

      Protective immunity against viral nervous necrosis (VNN) in brown-marbled grouper (Epinephelus fuscogutattus) following vaccination with inactivated betanodavirus 

      R Pakingking Jr., NB Bautista, EG de Jesus-Ayson & O Reyes - Fish and Shellfish Immunology, 2010 - Academic Press
      Viral nervous necrosis (VNN) caused by betanodaviruses has been recently implicated in serious mortalities of groupers in the grow-out culture system. A safe and effective vaccine against this disease is urgently needed. This study demonstrates that a single intramuscular vaccination with formalin-inactivated Philippine strain of piscine betanodavirus (genotype: redspotted grouper nervous necrosis virus; RGNNV) induces potent immune responses and substantial protective immunity against an intramuscular challenge with the homologous virus in brown-marbled grouper, Epinephelus fuscogutattus, a highly susceptible marine fish species to VNN. Seroneutralization assay conducted on sera of vaccinated fish revealed the occurrence of substantial neutralizing-antibody titers from Days 15 (mean titer 1:800) to 190 (1:400) with the highest titer observed at Day 60 post-vaccination (1:5120). When vaccinated fish were challenged with the homologous virus at Days 15, 30 and 75 post-vaccination, significantly higher survival rates were obtained in these fish compared with their corresponding controls (L-15 injected fish). Abrogation of virus multiplication in all vaccinated survivors was indicated by undetectable virus titers in the brains and kidneys paralleled by significantly high levels of neutralizing antibodies in the sera of these fish. Consecutively, replicates of vaccinated fish that survived betanodavirus challenge at Days 15 and 75 post-vaccination were maintained in flow-through aquaria and rechallenged with the homologous virus 3 and 5 months later, respectively. A significant drop in neutralizing-antibody titers of 3 and 8 folds, respectively, were observed in the sera of Days 15 and 75 post-vaccinated fish assayed before the virus rechallenge. Interestingly, reversion in the levels of neutralizing antibodies to significantly high levels (8–15 folds) were noted in these fish after the virus rechallenge. Taken together, our current data clearly demonstrate that a single administration of the inactivated Philippine strain of betanodavirus vaccine can effectively mount a specific anamnestic response and concomitant long-term protection against VNN in grouper at the grow-out culture system.
    • Article

      Purification and properties of a non-stereospecific dehalogenase enzyme E (DehE) from Methylobacterium sp. HJ1 

      NH Jing, FH Sulaiman, RA Wahab, RV Pakingking Jr., NAA Rashid & F Huyop - African Journal of Microbiology Research, 2008 - Academic Journals
      The bacterial isolate HJ1, which was identified as a Methylobacterium sp., grew on 2, 2-dichloropropionic acid as the sole carbon source and produced a 2-haloalkanoic acid hydrolytic dehalogenase. This non-stereospecific dehalogenase E (DehE) catalysed the hydrolytic dechlorination of 2, 2-dichloropropionic acid and D, L-2-chloropropionic acid to produce pyruvate and lactate, respectively. The enzyme was purified to homogeneity and characterized. The molecular weight was 36 kDa by SDS-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, suggesting that the enzyme is a protein dimer. The purified enzyme was only inhibited by HgSO4 and was non-stereospecific to haloalkanoic acids. The Km value for the hydrolysis of 2, 2-dichloropropionic acid was 0.25 mM. The enzyme removes chloride present on the α-position, but not on the β-position, of a number 2-carbon alkanoic acids.
    • Article

      Quantitative and qualitative analyses of the bacterial microbiota of tilapia (Oreochromis niloticus) cultured in earthen ponds in the Philippines 

      R Pakingking Jr., P Palma & R Usero - World Journal of Microbiology and Biotechnology, 2015 - Springer Verlag
      The quantity and composition of the bacterial microbiota in the rearing water, sediment, gills and intestines of tilapia Oreochromis niloticus collected every 2 weeks from Day 30 to Day 120 after stocking for grow-out culture in 6 earthen brackish water ponds in the Philippines were examined. The total heterotrophic aerobic bacterial counts obtained in the water, sediment, gills and intestines of tilapia ranged from 103 to 104 c.f.u. ml−1, 103–105, 105–107 and 104–107 c.f.u. g−1, respectively. In terms of composition, a total of 20 bacterial genera and 31 species were identified with the preponderance of gram-negative bacteria constituting 84% of all bacterial isolates examined. Aeromonas hydrophila, Bacillus spp., Plesiomonas shigelloides, Shewanella putrefaciens, Pseudomonas fluorescens, Staphylococcus spp. and Vibrio cholerae were the dominant bacteria identified in the gills and intestine of tilapia. These bacteria also dominated in the pond sediment and rearing water, except for the nil isolation of S. putrefaciens and V. cholerae in the water samples examined, indicating that resident bacteria in the pond water and sediment congruently typify the composition of bacterial microbiota in the gills and intestine of tilapia which under stressful conditions may propel the ascendance of disease epizootics.
    • Article

      Screening of vibriosis in Asian seabass, Lates calcarifer using loop-mediated isothermal amplification (LAMP) assay 

      CMA Caipang, RV Pakingking Jr. & MJS Apines-Amar - Human & Veterinary Medicine, 2012 - Bioflux Society
      The aim of this study was to standardize a loop-mediated isothermal amplification (LAMP) assay for the detection of Vibrio harveyi , the causative agent of vibriosis in Asian seabass, Lates calcarifer . The dnaJ gene of the bacterial pathogen was used as the target gene for the LAMP assay. It was optimized at an incubation time of 1 h at 63°C. The assay was highly specific for V. harveyi and did not cross-react with other bacterial pathogens offish. However, the assay was able to detect V. harveyi that was isolated from infected shrimps. The limit of detection of the LAMP assay was 40 pg of DNA mL-1 or 40 fg of the genomic DNA per LAMP reaction and was 10 times more sensitive than conventional PCR in detecting the bacterial pathogen from infected samples. The LAMP products can be quantified spectrophotometrically using hydroxynaphthol blue (HNB) dye and showed positive correlation with the amount of the pathogen. These results demonstrated that LAMP is a simple and sensitive detection technique that has potential application for routine diagnosis of vibrosis caused by V. harveyi in Asian seabass and other aquatic species.
    • Article

      Seasonal changes and coliform load of Jalaur river, province of Iloilo, Panay island, Philippines 

      I Lantin-Olaguer, SA Pedrajas-Mendoza, R Pakingking & A Yamamoto - Silliman Journal, 2010 - Silliman University
      Determination of the presence/absence of coliforms as the pollution indicator bacteria, total coliform count (TCC) and fecal coliform counts specifically Echerichia coli were carried out on specific sampling points in Jalaur River namely: Banban Pequeño (upstream), Calinog, Moroboro, Dingle, Passi near Sugar Central Mill and National Power Corporation (NPC) (midstream), and Nabitasan, Leganes (downstream) during the wet and dry months. Samples were analyzed using a defined technology, the Colilert® method, and its accuracy was verified with the conventional method (APHA Standard Methods).

      Results showed that coliforms were present in all sampling sites. In Calinog, total coliform count (TCC) was high (900 x101 MPN/100 ml) in July. Increased coliform abundance was associated with high rainfall due to animal wastes that were carried by runoffs. In September, Passi, near NPC, TCC and E. coli counts were 640 x101 and 630 x 101 MPN/100 ml, respectively. Reduction in counts in January, February and March was observed. Sediments in Passi near Sugar Central Mill during milling time in February showed a high TCC of 116 x 104 MPN/100 ml which was attributed to reduced current and disturbance, and silty loam sediments texture that favored bacterial adsorption to sediments. Dingle site exhibited a domination of other coliforms over E. coli in January and March. In Leganes, highest TCC of 551 x 101 MPN/100 ml was obtained in September. Coliform loads varied by season which was influenced by the availability of the nutrients and tolerance range to physical and chemical factors in the environment. Variability of the resultant interaction can also be attributed to climate changes such as extreme weather events—El niño phenomenon and increased nutrient loadings during heavy rains hence, increased coliform concentration in the river. The presence of coliforms in Jalaur River is indicative of contamination that can be aggravated by climate changes and implies that a potential health risk associated with pathogens causing water-borne diseases is present.
    • magazineArticle

      Supporting ASEAN good aquaculture practices: Preventing the spread of trans-boundary aquatic animal diseases 

      RV Pakingking Jr. & EG de Jesus-Ayson - Fish for the People, 2016 - SEAFDEC Secretariat
      The FAO Fishery Statistics had indicated that Asia is the top producer of fish and fishery products from both capture fisheries and aquaculture. Specifically, Southeast Asia had contributed 9-31% of the total aquaculture production in Asia from 1950 to 2014 with Indonesia and the Philippines accounting for the most at 23-63% and 10-45% of the total, respectively. Aquaculture has been viewed as a solution to the growing concern on food security issues as well as for the socio-economic stability of many countries in Southeast Asia. For such reason, aquaculture operations are being intensified to compensate for the declining production from capture fisheries and in order to nail the gap between supply and demand for fish and fishery products in the world. With intensification, aquaculture production has already overtaken the contribution of capture fisheries to the world’s total fisheries production. However, concerns on the safety and quality of aquaculture products have been raised as result of intensified fish farming operations. Added to such concern is the irresponsible introduction of aquatic species for aquaculture that serve as carriers of pathogens. As a result, a large number of infectious aquatic diseases have emerged threatening the sustainability of aquaculture in the Southeast Asian region. In an effort to address the emergence of transboundary diseases in the region, the Aquaculture Department of SEAFDEC (SEAFDEC/AQD) launched a program on Healthy and Wholesome Aquaculture which includes as one of its main objectives, the need to continue improving aquaculture production through innovations in fish health management.
    • Article

      Temporal changes in innate immunity parameters, epinecidin gene expression, and mortality in orange-spotted grouper, Epinephelus coioides experimentally infected with a fish pathogen, Vibrio harveyi JML1 

      EC Amar, JP Faisan Jr., MJS Apines-Amar & RV Pakingking Jr. - Fish and Shellfish Immunology, 2017 - Elsevier
      Changes in innate immunity parameters and epinecidin mRNA transcript levels were examined to characterize the non-specific immune response of E. coioides to pathogenic V. harveyi JML1 isolated from affected cage-cultured fish. After fish had been injected with bacteria at a dose causing 30% mortality, blood and tissue samples were collected at 0, 6, 12, 24, 48, 72, 96, 120, and 240 h post-infection (hpi) for assessment of indices such as the oxidative burst (OB) and phagocytic index (PI) of head kidney cells, and lysozyme activity (LYS) and total immunoglobulin (Total Ig) levels of the plasma. The epinecidin mRNA transcript levels (EGE) from skin, gills, liver, kidney, and spleen tissues were also determined by gelbased RT-PCR. Lastly, daily mortality (DM), liver total bacterial load (TBC), and presumptive Vibrio count (TVC) were monitored up to 240 hpi. The results revealed that bacteria proliferated rapidly in fish tissue, reaching peak densities at 24 hpi for both TBC and TVC but was on a downward trend thereafter. The pattern in fish mortality closely correlated with TBC and TVC. Total Ig, OB, and PI in E. coioides were suppressed in the early part of infection when V. harveyi load was high but recovered and later increased as bacterial density declined. LYS and EGE were consistently high and their activities were not hampered by bacterial infection. The study demonstrated that V. harveyi JML1 interacts with E. coioides by transiently inhibiting some immune parameters resulting in mortalities. However, consistently high LYS, upregulated EGE, and resurgent PI, OB and Total Ig conferred resistance and subsequent recovery in the fish. The study provides new insights on the interaction between E. coioides and V. harveyi JML1 that can aid in formulating health management strategies for groupers. Further studies on prophylactic interventions to enhance the innate immune response in grouper during infection with V. harveyi JML1 are suggested.