Genomic Cloning and Sequence Analysis of Trypanosoma brucei rhodesiense gene Encoding Putative N-glycosylation Enzyme

Background:  : : is a haemoflagellate parasite of zoonotic significance. Aside from its public health importance, this parasite subspecies gained notoriety because of their effective system to circumvent the immune response of vertebrate host. The parasite cell surface is covered with millions of VSG dimers, which serve as an almost infinite repertoire of biomolecules needed for evasion of host immune system. Around two decades ago, it was resolved that all trypanosome VSG is associated with one or more N-linked oligosaccharides, with a range of structures including high mannose and complex types. This complex process of protein modification known as N-linked glycosylation is catalyzed by oligosaccharyl transferase (OST). In general, the incorporation of glycan structures can alter protein’s antigenic properties and recently it was established that glycan molecules associated with VSG were found to be important in several aspects of trypanosome-host interaction, especially during parasite evasion of the host defense mechanisms. Therefore, our major interest is to clone and characterize the trypanosome OST. Material, Methods and Results: The template genomic DNA for PCR amplification was extracted as described previously. In an attempt to clone Trypanosoma brucei rhodesiense putative oligosaccharyl transferase, an amplicon of ~2000 bp was obtained having an open reading frame of 2057 bp and deduced primary structure composed of 685 amino acid residues (TbrOST II). Comparison of TbrOST II ORF with annotated putative oligosaccharyl transferase in the genome of other organisms revealed sequence identity to other kinetoplastid. TbrOST II had high nucleotide (Ns) and amino acid (As) sequence similarity with the genomes of T. brucei gambiense (Ns:99%; As:78%) and T. brucei (Ns:95-98%; As:77%-98%). There was also significant nucleotide and amino acid sequence identity in the genomes of T. cruzi (Ns:74%; As:63%), Leishmania infantum (Ns:70-83%; As:46-57%), L. braziliensis (Ns:69-81%; As:46-55%) and L. major (Ns:69-80%; As:46-57%). Sequence similarity (71-77%) from other origins was also exhibited. The nucleotide sequence alignments and analysis were performed using the Oxford University Mac Vector 6.5 sequence analysis software and CLC Workbench 5.6 software. Discussion: The nucleotide BLAST results indicate that sequence identity is higher between species of the same genus rather than of the same family. It is known that T. brucei, T. gambiense and T. rhodesiense are members of the Brucei-complex or Brucei group. Although T. brucei brucei has more similarities with T. brucei rhodesiense than T. brucei gambiense, these parasites are morphologically indistinguishable. This is the probable reason why high sequence identity was displayed by other subspecies of the Brucei group. In addition, the high percent identity possessed by TbrOST II with other trypansomatids agrees with the evolutionarily conserved characteristics of the established OST. The DNA sequence data of TbrOST II showing similar sequences in the genome of other organisms further corroborate the previous reports regarding the ubiquitous nature of OST in other life forms. Based on the size of the amplicon and significant percentage of nucleotide and amino acid sequence identity to homologues within the genome of related species and various organisms, the results strongly indicate that TbrOST II is a trypanosome oligosaccharyl transferase gene candidate that should be fully characterized and subjected to functional genomic studies. The study reports the molecular cloning and sequencing of a potential oligosaccharyl transferase gene in T. brucei rhodesiense (TbrOST II). The sequence data has been deposited in the GenBank with accession number GU475126.Trypanosoma brucei rhodesiens

Trypanosoma evansi ima gen sličan genu za oligosaharil-transferazu klona I protozoona Trypanosoma brucei rhodesiense.

Recent data has shown that there are strong indications that the putative oligosaccharyl transferase genes from Trypanosoma brucei rhodesiense were conserved within the family Trypanosomatidae. Based on these findings, the study endeavored to determine if Trypanosoma evansi also possessed putative oligosaccharyl transferase (OST) clone I previously documented in Trypanosoma brucei rhodesiense. Using the DNA hybridization method (Southern blot analyses), genomic DNAs of Trypanosoma brucei rhodesiense and Trypanosoma evansi were processed using the same set of restriction enzymes and subsequently hybridized by the same set of DNA probes designed from the reported nucleotide sequence of Trypanosoma brucei rhodesiense putative oligosaccharyl transferase clone I. The results exhibited that Trypanosoma evansi also contains a gene similar to the reported Trypanosoma brucei rhodesiense putative OST gene clone I, as shown by the successful hybridization of the DNA probes to their complementary nucleotide sequences in the genome of the Trypanosoma evansi species. In addition, the data also showed that Trypanosoma brucei rhodesiense and Trypanosoma evansi genomes shared some common restriction sites and loci within the genome of each individual parasite species.Nedavna istraživanja pokazala su da su geni za oligosaharil transferazu protozoona Trypanosoma brucei rhodesiense vrlo dobro sačuvani unutar porodice Trypanosomatidae. Cilj istraživanja bio je otkriti je li ista pojava karakteristična za gen za oligosaharil transferazu klona I protozoona Trypanosoma evansi. Genomske DNA vrste Trypanosoma brucei rhodesiense i vrste Trypanosoma evansi bile su pretražene hibridizacijom DNA (Southern Blot analizom) rabeći istu skupinu restrikcijskih enzima kao i iste probe za hibridizaciju DNA pripravljene na temelju objavljenog slijeda nukleotida za oligosaharil-transferazu klona I protozoona Trypanosoma brucei rhodesiense. Rezultati su pokazali da Trypanosoma evansi također sadrži gen koji je vrlo sličan genu za oligosaharil-transferazu protozoona Trypanosoma brucei rhodesiense, što je dokazano uspješnom hibridizacijom DNA proba s komplementarnim nukleotidnim slijedovima u genomu vrste Trypanosoma evansi. Istraživanje je pokazalo da Trypanosoma brucei rhodesiense i Trypanosoma evansi dijele i zajednička restrikcijska mjesta

Izdvajanje bakterije Escherichia coli O157:H7 i njezin dokaz lančanom reakcijom polimerazom u crijevima filipinskih šišmiša.

It is currently reported that bats in the Philippines harbor bacterial organism (Salmonella spp.) with pathogenic potential. The paper describes the conventional isolation and polymerase chain reaction (PCR) assay for the detection of another bacterium, Escherichia coli, from a sample population of 56 apparently healthy bats collected from Laguna and Quezon City, Philippines. Nineteen of the samples were positive for E. coli using the conventional method of isolation, while PCR molecularly detected the bacteria in 15 samples. The presence of hemolysin among the isolates was not observed. The isolates were subjected to E. coli O157:H7 serotype detection using the latex agglutination test and another PCR assay specific for this serotype. The data revealed that none of the isolates was positive for E. coli O157:H7 using serological and molecular diagnostic methods, which indicates that bats from Laguna and Quezon City, Philippines were not carriers of the pathogenic strain, E. coli O157:H7. The study also presents the first local report of conventional isolation and molecular detection of E. coli from Philippine bats.Poznato je da šišmiši na Filipinima mogu biti nositelji potencijalno patogenih bakterija, npr. salmonela. U radu je opisano izdvajanje i dokaz lančanom reakcijom polimerazom bakterije Escherichia coli iz 56 naizgled zdravih šišmiša uhvaćenih na području Laguna i Quezon City na Filipinima. E. coli bila je izdvojena iz 19 uzoraka, dok je lančanom reakcijom polimerazom ona bila dokazana u 15 uzoraka. Izolati nisu tvorili hemolizu, a lateks aglutinacija i specifični PCR rabljeni su za dokaz serovara O157:H7 bakterije E. coli. Nijedan izolat nije pripadao serovaru O157:H7, na osnovi čega se može zaključiti da šišmiši na području Laguna i Quezon City na Filipinima nisu nositelji patogenog soja E. coli O157:H7. Istraživanje je ujedno prvi dokaz o izdvajanju i molekularnoj identifikaciji bakterije E. coli u šišmiša na Filipinima

Histopathology of protozoal infection in animals: a retrospective study at the University of Philippines College of Veterinary Medicine (1972-2010)

The authors describe the first parasitological survey of protozoal infections on tissue slide sections of field cases processed at the histopathology laboratory of the College of Veterinary Medicine (CVM) at the University of the Philippines Los Baños (UPLB). Over 80% of the field cases were from Region 4 (CALABARZON) and the rest were equally distributed from other areas of the Philippines, namely: Region 2 (Cagayan Valley), Metropolitan Manila (National Capital Region), Region III (Central Luzon) and Region VI (Western Visayas). Histopathological analyses of tissue sections from 51 archived cases (1972-2010) of parasitic aetiology were performed. Microscopic examination of a total of 286 histopathological slides revealed the presence of several protozoa, including sarcosporidiosis, hepatic coccidiosis, intestinal coccidiosis, balantidiosis and leucocyto-zoonosis. In addition, the finding of Balantidium and Sarcocystis may have zoonotic implications and can therefore be used as markers of public health importance

Dokaz nametnika Trypanosoma evansi lančanom reakcijom polimerazom u goveda na području Quirino na Filipinima

Trypanosoma evansi is a protozoan parasite which ubiquitously infects livestock in the Philippines. The study depicts an initial report of livestock trypanosomes in cattle from the province of Quirino, Philippines using the polymerase chain reaction (PCR) method. Out of 246 field blood samples from apparently healthy cattle collected from five municipalities of Quirino Province, Philippines, 3/246 (1.22%) were established positive for T. evansi using PCR. The positive animals identified were from the municipality of Saguday (3/42; 7.14% prevalence). These were composed of one female and two males species and belonged to variable age groups (1.1-2 yrs, 2.1-3 yrs and >3 yrs). In addition, the data on the physical examination of the sampled animals using the nine-point scale body condition scoring system showed that the top three body condition scores (BCS) were 3[Very thin, (107/246; 43.50%)], 4[Thin, (75/246; 30.50%)] and 2[Emaciated, (46/246; 18.70%)]. Furthermore, the data showed that majority of the animals in Diffun and Saguday were thin (BCS 4) while most of the animals in Cabarroguis and Maddela and Aglipay were classified under BCS 3(Very thin) and BCS 2(Emaciated) respectively. Consequently, all the Trypanosoma evansi positive animals appeared thin (BCS= 4) and were from the municipality of Saguday. No parasites were observed after blood parasite examination (BPE). The study provides information on the first report of T. evansi cases in cattle from Quirino Province located in Region 2 of the Philippines. Consequently the data also presents the first molecular detection of T. evansi infection in cattle in the province of Quirino, Philippines.Trypanosoma evansi posvudašnji je protozoon koji invadira stoku na Filipinima. To je prvo izvješće o tripanosomozi goveda na području Quirino na temelju nalaza dobivenih lančanom reakcijom polimerazom. Ukupno je 246 uzoraka krvi bilo sakupljeno od naizgled zdravih goveda u pet općina na području Quirino. Prisutnost T. evansi dokazana u 3 (1,22%) životinje, u jedne ženke i dva mužjaka različite dobi (1,1-2 godine, 2,1-3 godine i više od 3 godine) u općini Saguday (3/42; prevalencija od 7,14% ). U svih životinja bila je određena i tjelesna kondicija prema sustavu bodovanja od 1 do 9. Pregledom svih životinja ustanovljena su bila tri najčešća stupnja kondicije: 3 [vrlo mršava, (107/246; 43,50%)], 4 [mršava, (75/246; 30,50%)] i 2 [kahektična, (46/246; 18,70%)]. Goveda na području naselja Diffun i Saguday bila su mršava dok su goveda na području Cabarroguis, Maddela i Aglipay bila vrlo mršava i kahektična. Sve su pozitivne životinje u općini Saguday bile mršave. U goveda pozitivnih lančanom reakcijom polimerazom nisu bili dokazani paraziti u krvi. Rezultati ovog istraživanja predstavljaju i izvješće o prvom dokazu protozoona Trypanosoma evansi na području Quirino na Filipinima