Molecular Characterization of Vibrio spp. in Shellfish using Multilocus Sequence Analysis

Fish and shellfish are the second largest source of protein for man after meat products and in some countries, such as Japan, constitute the main source of protein. In recent years, indigenous marine bacteria were responsible for 20% of all diseases and 99% of fatalities associated with the consumption of fishery products (Cozzi and Ciccaglioni, 2005). Among these, the main causes of diseases are some species of Vibrionaceae, which can cause gastroenteritis, especially after the consumption of fish products, raw or undercooked, from temperate and warm Seas. Vibrio is a very diverse genus responsible of different human and animal diseases. The accurate identification of Vibrio spp. is very important to assess the risks in regard to public health and diseases of aquatic organisms. Thus, analyses of population structure for a reliable bacteria characterization in different ecological environments are necessary. In particular, sequence based identification methods are preferable over classical biochemical approaches. In this study, a Multilocus Sequence Analysis scheme was developed on the basis of four housekeeping genes (gyrB, pyrH, recA and atpA) applied to 3 set of Vibrio strains (154 isolates from mollusks in 2007; 92 isolates from crustacean and 22 isolates form mollusks in 2011 ) and 29 reference strains. Concatenated sequences were used for phylogenetic and population analyses and the results were compared with biochemical identification tests (Alsina’s scheme). The phylogeny provided a good clustering, showing 15 clusters and 6 single strains in the first set of strains; 10 clusters and 4 singletons in second set; and 4 clusters and 4 singletons in the third set of strains. The population analysis highlighted 17 subpopulations in first set and 12 subpopulations in second set of Vibrio strains that were well supported by phylogeny with few exceptions. Overestimations of risk due to biochemical identification have been found for V. parahaemolyticus and V. vulnificus and no V. cholerae strains were identified. The false negative results of Alsina’s scheme need to be considered as it might represent a potential public health risk. These findings highlight the need of a rapid and robust identification of shellfish associated foodborne Vibrio spp. and, in addition, the connection of environmental information to genetic data could enhance the Vibrio spp. characterization. Second part of the study gave special emphasis on the species Vibrio parahaemolyticus, a potential emerging pathogen in the North Adriatic Sea. Pathogenic strains of V. parahaemolyticus represent one of the main causes of foodborne gastroenteritis, especially in Asia and USA (Su and Liu, 2007). The study examined 160 strains isolated from 43 edible mollusks sampled between January and October 2011, identified biochemically as Vibrio parahaemolyticus in the Food Microbiology laboratory of Istituto Zooprofilattico (IZSVe). The strains were characterized for the presence of genes typical for the species Vibrio parahaemolyticus (toxR and tlh) in order to confirm the biochemical identification and virulence genes (tdh and trh). Dubious or misidentified strains were subjected to MLSA (Multilocus Sequence Analysis) by evaluating the sequence of 4 housekeeping genes. Finally, 102 Vibrio parahaemolyticus strains were analyzed by the MLST protocol: portions of 7 genes (dnaE, gyrB, recA, dtdS, pntA, pyrC and tnaA) were sequenced and concatenated. With the obtained MLST information phylogenetic analyses were performed to determine the relationships between the different strains isolated in this study and secondly, any links with worldwide isolates. All strains of V. parahaemolyticus were found positive for toxR and tlh, no strain was tdh positive, while 6 strains had the positive reaction for trh gene. 72 non-redundant (63 new) STs were identified. A total of 54 clonal groups were highlighted, in which 17 are clonal complex. Two distinct populations of V. parahaemolyticus were marked by phylogenetic, structure and recombination analyses. The main result is that despite the high percentage of positive samples for V. parahaemolyticus, only a few strains were potentially pathogenic for humans. However, some possible genetic relationships with strains can emerge from a comparative study with the STs in the world database. The characterization could help to identify suspect genotypes and thus clarify the dynamics of the spread of potentially pathogenic strains

FAC-PIN: An efficient and fast agglomerative clustering algorithm for protein interaction networks to predict protein complexes and functional modules

Proteins are known to interact with each other to perform specific living organism functions by forming functional modules or protein complexes. Many community detection methods have been devised for the discovery of functional modules or protein complexes in protein interaction networks. One common problem in current agglomerative community detection approaches is that vertices with just one neighbor are often classified as separated clusters, which does not make sense for module or complex identification. In this thesis, we propose a new agglomerative algorithm, FAC-PIN, based on a local premetric of relative vertex-to-vertex clustering value. Our proposed FAC-PIN method is applied to PINs from different species for validating functional modules and protein complexes generated from FAC-PIN with experimentally verified functional modules and complexes respectively. The preliminary computational results show that FAC-PIN can discover functional modules and protein complexes from PINs more accurately. As well as we have also compared the computational times for different species with HC-PIN and CNM algorithms. Our algorithm outperforms two algorithms. Our FAC-PIN algorithm is faster and accurate algorithm which is the current state-of-the-art agglomerative approach to complex prediction and functional module identification

GENETIC DIVERSITY BY RAPD IN FOUR POPULATIONS OF ROHU Labeo rohita

Genetička raznolikost među četiri geografski udaljene populacije indijskog šarana (Labeo rohita) - rohu Khulna, rohu India, rohu Faridpur i rohu Barisal - određena je pomoću RAPD markera. Uzorak na gelu je pokazao da je populacija rohu India genetski različita od triju ostalih. Ukupno je pronađeno 87 crtica u četiri rohu populacije pri čemu je 35 crtica bilo polimorfno, što ukazuje na 48,38% polimorfizma s prosjekom od 12 crtica po početnici. Dužina amplificiranih fragmenata DNK kretala se u rasponu od 400 do 1250 bp. Osam jedinstvenih crtica je uočeno u četiri populacije od kojih je šest pronađeno u rohu India populaciji. Genetska udaljenost je bila najviša (0,7221) i genetska identičnost je bila najniža (0,4857) između populacija rohu India i rohu Barisal. Među tri lokalne populacije najniža genetska udaljenost i najviša genetska identičnost utvrđena je između populacija rohu Khulna i rohu Faridpur. UPGMA filogenetsko stablo odijelilo je četiri populacije indijskog šarana u tri glavna odjeljka - C1, C2 i C3. Rohu Indija nalazi se na potpuno drugačijem odjeljku C3, rohu Khulna je smještena u C2, a ostale dvije populacije u C1 odjeljak.Genetic diversity in four geographically distinct rohu (Labeo rohita) populations such as rohu Khulna, rohu India, rohu Faridpur and rohu Barisal was determined by RAPD. The banding pattern showed that rohu India was genetically different from the rest three. A total of 87 bands were found in four rohu populations where 35 bands were polymorphic, indicating 48.38% polymorphisms with an average of 12 bands per primer. The molecular size of amplified DNA fragments ranged from 400 to 1250 bp. Eight unique bands were observed in the four populations of which six were found in rohu Indian population. The genetic distance was highest (0.7221) and genetic identity was lowest (0.4857) between rohu India and rohu Barisal populations. Among three local varieties, the lowest genetic distance and highest genetic identity were found between rohu Khulna and rohu Faridpur populations. The UPGMA dendrogram segregated four populations of rohu into three major clusters - C1, C2 and C3. Rohu India was positioned at a fully different cluster - C3, rohu Khulna was placed in C2 and the rest two populations in C1

Ethnomedicine in Sabah : past trends and future perspectives

Introduction: The objective of this presentation is to carry out a review of the historical status and possible future outlook of the practice of ethnomedicine in the State of Sabah. As in many parts of the world, there are many types of traditional medicines that have been used for hundreds of years among the various native cultural groups in Sabah. Methods: A literature survey was conducted with the aid of online database, and text books using keywords such as the current as well as previous names of Sabah and various related terminology such as ethnomedicine, CAM, complementary and alternative medicine, TCM, traditional and complementary medicine, herbalism, reflexology, spiritualism, ethnobiology ethnology, ethnoanthropology, biological activities, ethnobotanical uses, medicinal prop-erties, toxicity studies, etc. The status of ethnomedicine in Sabah is largely unknown. Results: Based on a review of previous literature, the study discovered that there is limited empirical documentation on the use of ethnomedicine in Sabah. In this preliminary review, a total number of 25 publications were recorded in the databases for the period up to 2019. There is still a need to realise the full potential of the biocultural diversity of Sabah, and that there is a strong need to focus more on socially relevant research. Ethnomedicine in Sabah consists mainly of herbalism, ma-nipulative practices and spiritualism. Plants can be said to be the central player in race-based traditional medicine. While data on ethnomedicine may be based on original field research, several are primarily derivative, drawing as they do from earlier published accounts and, importantly, anecdotes. Traditional massage and other manipula-tive-practices, based on manipulation and movement of body parts, are also popular because of their simplicity and apparent immediate relief that can be experienced by the patient or client. Spiritualism, which may be considered as a peculiar dimension of the mind-body medicine subset of traditional medicine, continues to have its adherents among the natives of Sabah. Conclusion: List and catalogs and sociological and ethnohistorical data were the fa-voured perspective of research in the past; but future evolution of ethnomedical studies need to address some of the debates related to biological, medical and social sciences . Future research should include systematic inves-tigation into efficacy and risk besides the possible incorporation of some parts of ethnomedicine into the existing healthcare services. Ethnomedicine occupies a distinct place in healthcare in the world in general, and Sabah in particular

Seasonal variations in nutritional profile of the freshwater mud eel, Monopterus cuchia (Hamilton, 1822)

Seasonal variations of proximate compositions, amino acids, and fatty acids contents of Monopterus cuchia were studied for the first time in Bangladesh. The fat and fatty acid, protein and amino acid contents showed a significant seasonal dependency. Lipid contents showed the wider variation than the protein contents. Among the 14 recorded amino acids, the primary amino acids were lysine, glutamic acid, arginine, glycine and aspartic acid. The ratio of essential and non-essential amino acids was higher in the post-monsoon season (0.69) than pre-monsoon (0.68) and monsoon season (0.68) and did not show seasonal discrepancies. The amount of fatty acids were found in order of saturated fatty acids (SFA) > mono unsaturated fatty acids (MUFA) > polyunsaturated fatty acids (PUFA). The predominant fatty acids were palmitic and stearic acids of SFAs, oleic and palmitoleic acid of MUFAs and linoleic and α linolenic acids of PUFAs group. The combined value of EPA+DHA varied from 1.44–5.34% depending on the season, with the highest in monsoon season. However, the n-6/n-3 ratios were notably lower (0.58–2.51) throughout the season than the greatest esteem of 4. Therefore, M. cuchia may be consumed as healthy as safe food with reference to n-6/n-3 ratio, irrespective of catching seasons

Prevalence and antibiotic susceptibility of Aeromonas hydrophila isolated from freshwater fishes

Aeromonas hydrophila is an opportunistic microorganism. It is a secondary biological agent that contributes to the occurrence of fish diseases and its deterioration. This research was undertaken to determine the prevalence of A. hydrophila in some freshwater fishes collected from three different fish markets of Dhaka City and to test their antibiotic susceptibility. Total bacterial count and total aeromonas on different aeromonas selective media were enumerated using serial dilution technique. Bacterial isolates were characterized to identify A. hydrophila using biochemical tests and with comparison to reference strain (ATCC 7966). The lowest Aeromonas count was detected to be 2.83±0.40×102 cfu/g in Anabas testudineus and the highest was 1.03±0.153×103 cfu/g in Oreochromis mossambicus. On market basis highest aeromonas count was found in Anando Bazar (8.10±1.09×102 cfu/g) and lowest in Hatirpool Bazar (5.63±0.90×102 cfu/g) with no significant difference. Maximum susceptibility to amikacin and gentamicin was observed whereas all of the isolates were found resistant to a commonly used antibiotic amoxycillin. The obtained results point that antimicrobial susceptibility was more or less similar regardless of the origin of the samples collected. All the fishes investigated in this study contained A. hydrophila in their different organs

A comparison of the ecology of Deschampsia Caespitosa and Dactylis Glomerata with special reference to their water relations

Imperial Users onl

Molecular Characterization of Vibrio spp. in Shellfish using Multilocus Sequence Analysis

Fish and shellfish are the second largest source of protein for man after meat products and in some countries, such as Japan, constitute the main source of protein. In recent years, indigenous marine bacteria were responsible for 20% of all diseases and 99% of fatalities associated with the consumption of fishery products (Cozzi and Ciccaglioni, 2005). Among these, the main causes of diseases are some species of Vibrionaceae, which can cause gastroenteritis, especially after the consumption of fish products, raw or undercooked, from temperate and warm Seas. Vibrio is a very diverse genus responsible of different human and animal diseases. The accurate identification of Vibrio spp. is very important to assess the risks in regard to public health and diseases of aquatic organisms. Thus, analyses of population structure for a reliable bacteria characterization in different ecological environments are necessary. In particular, sequence based identification methods are preferable over classical biochemical approaches. In this study, a Multilocus Sequence Analysis scheme was developed on the basis of four housekeeping genes (gyrB, pyrH, recA and atpA) applied to 3 set of Vibrio strains (154 isolates from mollusks in 2007; 92 isolates from crustacean and 22 isolates form mollusks in 2011 ) and 29 reference strains. Concatenated sequences were used for phylogenetic and population analyses and the results were compared with biochemical identification tests (Alsina’s scheme). The phylogeny provided a good clustering, showing 15 clusters and 6 single strains in the first set of strains; 10 clusters and 4 singletons in second set; and 4 clusters and 4 singletons in the third set of strains. The population analysis highlighted 17 subpopulations in first set and 12 subpopulations in second set of Vibrio strains that were well supported by phylogeny with few exceptions. Overestimations of risk due to biochemical identification have been found for V. parahaemolyticus and V. vulnificus and no V. cholerae strains were identified. The false negative results of Alsina’s scheme need to be considered as it might represent a potential public health risk. These findings highlight the need of a rapid and robust identification of shellfish associated foodborne Vibrio spp. and, in addition, the connection of environmental information to genetic data could enhance the Vibrio spp. characterization. Second part of the study gave special emphasis on the species Vibrio parahaemolyticus, a potential emerging pathogen in the North Adriatic Sea. Pathogenic strains of V. parahaemolyticus represent one of the main causes of foodborne gastroenteritis, especially in Asia and USA (Su and Liu, 2007). The study examined 160 strains isolated from 43 edible mollusks sampled between January and October 2011, identified biochemically as Vibrio parahaemolyticus in the Food Microbiology laboratory of Istituto Zooprofilattico (IZSVe). The strains were characterized for the presence of genes typical for the species Vibrio parahaemolyticus (toxR and tlh) in order to confirm the biochemical identification and virulence genes (tdh and trh). Dubious or misidentified strains were subjected to MLSA (Multilocus Sequence Analysis) by evaluating the sequence of 4 housekeeping genes. Finally, 102 Vibrio parahaemolyticus strains were analyzed by the MLST protocol: portions of 7 genes (dnaE, gyrB, recA, dtdS, pntA, pyrC and tnaA) were sequenced and concatenated. With the obtained MLST information phylogenetic analyses were performed to determine the relationships between the different strains isolated in this study and secondly, any links with worldwide isolates. All strains of V. parahaemolyticus were found positive for toxR and tlh, no strain was tdh positive, while 6 strains had the positive reaction for trh gene. 72 non-redundant (63 new) STs were identified. A total of 54 clonal groups were highlighted, in which 17 are clonal complex. Two distinct populations of V. parahaemolyticus were marked by phylogenetic, structure and recombination analyses. The main result is that despite the high percentage of positive samples for V. parahaemolyticus, only a few strains were potentially pathogenic for humans. However, some possible genetic relationships with strains can emerge from a comparative study with the STs in the world database. The characterization could help to identify suspect genotypes and thus clarify the dynamics of the spread of potentially pathogenic strains.I prodotti ittici sono la seconda fonte di proteine per l’alimentazione dell'uomo e in alcuni Paesi, quali il Giappone, ne costituiscono la principale fonte. Negli ultimi anni, i batteri marini della flora indigena sono risultati responsabili del 20% delle malattie nell’uomo e del 99% dei decessi derivati dal consumo dei prodotti della pesca. Tra questi, le principali cause di malattie sono da ascrivere ad alcune specie di Vibrionaceae in particolare al genere Vibrio, che possono causare gastroenteriti, soprattutto a seguito di consumo di prodotti crudi o poco cotti, provenienti da mari temperati e caldi. L'identificazione accurata dei batteri appartenenti al genere Vibrio risulta quindi molto importante per valutare i rischi in materia di salute pubblica e per l’identificazione puntuale delle malattie degli organismi acquatici. Risulta quindi necessario sviluppare ed applicare metodi affidabili che possano caratterizzare le specie di vibrioni residenti nei prodotti commercializzati (es. molluschi bivalivi e crostacei). In particolare, i metodi di identificazione basati sull’analisi delle sequenze geniche sono preferibili rispetto ai classici approcci biochimici. In questo studio è stato sviluppato uno schema MLSA Multilocus Sequence Analysis impiegando quattro geni housekeeping (gyrB, pyrH, recA e atpA), tale schema è stato valutato in 3 differenti data set di ceppi (154 isolati da molluschi nel 2007; 92 isolati di crostacei e 22 da molluschi isolati nel 2011) e 29 ceppi di riferimento e Type strain. I concatenameri sono stati utilizzati per le analisi filogenetiche e per gli studi di popolazione dei Vibrio isolati, confrontando al contempo i risultati dell’identificazione di specie con i test biochimici (schema di Alsina) applicati di routine all’identificazione dei Vibrioni. L’analisi della struttura di popolazione mediante il software STRUCTURE e l’analisi filogenetica risultano concordi nell’assegnazione dei principali taxa evidenziando una simile clusterizzazione dei gruppi in sottopopolazioni. Al contrario, il confronto tra la classificazione mediante MLSA e i test biochimici ha evidenziato varie discrepanze tra le quali una sovrastima di ceppi classificati come V. parahaemolyticus e V. vulnificus. Al contempo alcuni ceppi di V. parahaemolyticus sono risultati falsi negativi. Questi riscontri potrebbero indicare una limitazione dell’utilizzo delle prove biochimiche adottate di routine alla classificazione dei Vibrio potenzialmente patogeni per l’uomo e tale riscontro si riflette in un possibile rischio per la salute pubblica. La seconda parte dello studio ha considerato nel dettaglio la caratterizzazione molecolare di V. parahaemolyticus. Questo batterio è oggi un patogeno emergente derivato dal consumo di prodotti ittici, infatti ceppi patogeni di V. parahaemolyticus rappresentano una delle principali cause di gastroenterite di origine alimentare, in particolare in alcuni paesi dell’Asia e negli Stati Uniti. Questo batterio, a causa di mutamenti ambientali e delle abitudini dei consumatori (consumo di prodotti crudi provenienti da aree contaminate) potrebbe rappresentare una problematica igienico sanitaria anche nel Mare Adriatico settentrionale. In questa parte dello studio sono stati esaminati 160 ceppi isolati da 43 campioni di molluschi commestibili campionati tra gennaio e ottobre 2011 e identificati a livello biochimico dal laboratorio di microbiologia dell’Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe). I ceppi sono stati caratterizzati per la presenza dei marker genici specie specifici (toxR e tlh - Vibrio parahaemolyticus) per confermare l'identificazione biochimica e quindi dei geni per i fattori di virulenza (tdh e trh). I ceppi risultati di dubbia o errata identificazione sono stati sottoposti a MLSA (Multilocus Sequence Analysis) valutando la sequenza dei 4 geni housekeeping. Infine tutti i ceppi risultati Vibrio parahaemolyticus (n° 102) sono stati analizzati mediante il protocollo MLST (http://pubmlst.org/vparahaemolyticus/.). Lo schema prevede l’analisi di sequenza di 7 porzioni geniche (dnaE, gyrB, recA, dtdS, pntA, pyrC and tnaA). I concatenameri ottenuti sono stati utilizzati nelle analisi bioinformatiche di popolazione per determinare le relazioni tra i diversi ceppi isolati in questo studio e, in seconda battuta, per evidenziare eventuali collegamenti con ceppi isolati a livello mondiale. Per quanto concerne i fattori di virulenza tutti i ceppi di V. parahaemolyticus sono risultati tdh negativi, mentre 6 ceppi hanno presentato la positività per il gene trh. Nel complesso sono stati identificati 72 profili ST non ridondanti, 63 dei quali di nuova attribuzione rispetto al database on-line. L’analisi clonale dell’intero database ha evidenziato la presenza di 54 gruppi clonali dei quali 17 risultano essere ascritti entro un complesso clonale. Le analisi di popolazione nel loro complesso delineano la presenza di due gruppi principali di V. parahaemolyticus. Dallo studio emerge che, nonostante sia stata riscontrata un’alta percentuale di campioni positivi per V. parahaemolyticus, solo pochi ceppi risultano potenzialmente patogeni per l'uomo. Tuttavia, alcune possibili relazioni genetiche con ceppi isolati da casi di gastroenteriti in varie parti del mondo emergono dallo studio comparativo con il database on-line. La caratterizzazione molecolare potrebbe aiutare a individuare genotipi sospetti e quindi chiarire la dinamica della diffusione di ceppi potenzialmente patogeni