Untypable Strains Research Articles

Whole genome detection of rotavirus mixed infections in human, porcine and bovine samples co-infected with various rotavirus strains collected from sub-Saharan Africa

Group A rotaviruses (RVA) are among the main global causes of severe diarrhea in children under the age of 5years. Strain diversity, mixed infections and untypeable RVA strains are frequently reported in Africa. We analysed rotavirus-positive human stool samples (n=13) obtained from hospitalised children under the age of 5years who presented with acute gastroenteritis at sentinel hospital sites in six African countries, as well as bovine and porcine stool samples (n=1 each), to gain insights into rotavirus diversity and evolution. Polyacrylamide gel electrophoresis (PAGE) analysis and genotyping with G-(VP7) and P-specific (VP4) typing primers suggested that 13 of the 15 samples contained more than 11 segments and/or mixed G/P genotypes. Full-length amplicons for each segment were generated using RVA-specific primers and sequenced using the Ion Torrent and/or Illumina MiSeq next-generation sequencing platforms. Sequencing detected at least one segment in each sample for which duplicate sequences, often having distinct genotypes, existed. This supported and extended the PAGE and RT-PCR genotyping findings that suggested these samples were collected from individuals that had mixed rotavirus infections. The study reports the first porcine (MRC-DPRU1567) and bovine (MRC-DPRU3010) mixed infections. We also report a unique genome segment 9 (VP7), whose G9 genotype belongs to lineage VI and clusters with porcine reference strains. Previously, African G9 strains have all been in lineage III. Furthermore, additional RVA segments isolated from humans have a clear evolutionary relationship with porcine, bovine and ovine rotavirus sequences, indicating relatively recent interspecies transmission and reassortment. Thus, multiple RVA strains from sub-Saharan Africa are infecting mammalian hosts with unpredictable variations in their gene segment combinations. Whole-genome sequence analyses of mixed RVA strains underscore the considerable diversity of rotavirus sequences and genome segment combinations that result from a complex evolutionary history involving multiple host species.

Serovars and Drug-Resistance of Salmonella Strains Isolated from Domestic Chicken Meat in Tokyo (1992-2012)

A total of 477 Salmonella strains isolated from retail domestic chicken meat during 1992-2012 in Tokyo, were examined regarding their serovars and drug-resistance. These strains were detected in 469 (29.8%) of 1,576 samples. The detection rate in every two years was 10.1% to 46.3% of the range. Serological typing results showed that 477 strains were classified into 22 serovars excepting 2 untypable strains. Among them, S. Infantis (312 strains) was the most prevalent, followed by II O4: b: [e, n, x] (S. II Sofia) (71 strains), S. Hadar (20 strains), S. Typhimurium (20 strains), S. Manhattan (12 strains), S. Schwarzengrund (9 strains), S. Agona (7 strains), and other 15 serovars (24 strains). Results of the antibacterial drug susceptibility test for 477 strains revealed that 89.9% was resistant to some of the 12 drugs tested, and multidrug-resistant strains accounted for 90.2% among them. The frequencies of resistance to each drug were 81.8%; 77.8%, 45.5%, 33.3%, 11.3%, 9.6%, 2.9%, 0.6%, 0.6% and 0.2%, in order with high frequency, for SM, TC, KM, ST, NA, ABPC, CP, FOM, CTX and CAZ, respectively. None of the strains was resistant to NFLX or IPM. Three CTX-resistant strains were CTX-M type extended-spectrum β-lactamase (ESBL) producers, and the group of CTX-M type ESBL genes were CTX-M-2 group (2 strains) and CTX-M-9 group (1 strain). CAZ-resistant 1 strain was an ESBL producer, but the ESBL gene was not determined.

Quantitative and Qualitative Studies on Enterobacteriaceae in Ground Beef

120 packages of ground beef (40 each of fresh, frozen and frozen with Soya bean) were examined for the incidence of Enterobacteriaceae, Salmonella and E. coli. The incidence of Enterobacteriaceae was 100%, 75% and 87% in fresh, frozen and frozen with Soya ground beef respectively; and the mean counts were 6.3x104cfu/g, 1.4x102cfu/g and 1.6x103cfu/g respectively. E. coli, Klebsiella oxytoca, Serratia marcescens and Serratia liquefaiens could be isolated from fresh ground beef; Proteus vulgaries, E. coli and Enterobacter sakazakii was isolated from frozen ground beef, while in case of frozen ground beefwith Soya bean Klebsiella oxytoca, Proteus vulgaries, Citrobacter freundii, E. coli and Pantoea a gglomerans was isolated. The incidence of E. coli was 60%, 35%, 48% in fresh, frozen and frozen ground beef with Soya bean respectively. The incidence of Salmonellawas 0%, 10%, 15% in fresh, frozen and frozen ground beef with Soya bean respectively. E. coli strains were serologically identified into 20 O-typable strains and 37 O- untypable strains. The 20 O-typable strains were identified as O86, O119, O158 ,O145 and O115 .

Bacteriological studies on native and imported apparently healthy one day old chicks

One thousands and eighty one day old chicks were examined for isolation of different bacteria. Salmonella was detected in 11.7% in native chicks while 5.2% among imported chicks, E. coli was isolated from 17.6% in native chicks while 23.6% in imported, Staph aureus was present in 29.4% in native and 5.2% in imported chicks. Serological typing of Salmonella was Salmonella Norwich, Salmonella Wilhelburg in native chicks while in imported chicks were Salmonella Brancoster, Salmonella Sekondi II. E .coli sero-typing reveled O125, O153, O86a in native chicks while in imported chicks were O26, O78, O36, O15, O124, O169, O6, O28 and one un-typeable strain. Seven Staph aureus isolates five from native and two from imported. Antibiogram of isolated bacteria was done. All Salmonella strains were sensitive to Gentamycin while all E. coli strains were sensitive to Amoxicillin + Clavulanic acid and Nitrofurantoin. All Staph aureus isolates were sensitive to Amoxicillin + Clavulanic acid.

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: Predominance of P[8] genotype resembling human strains

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1–29.7%). This study revealed that age, management system and pig density significantly (p<0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811–1604nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4–91.3% and 95.1–96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6–100% to 92–100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6–91.7% and 89.3–96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

Molecular epidemiology and antibiotic resistance pattern of Enteropathogenic Escherichia coli isolated from bovines and their handlers in Jammu, India

The study was aimed to investigate the molecular epidemiology and antibiotic resistance pattern of Enteropathogenic Escherichia coli (EPEC) in bovines and their handlers in Jammu, India. A total of 173 samples comprising of 103 fecal samples from bovines (60 from cattle and 43 from buffaloes), 28 stools and 42 fingertip rinses from bovine handlers were collected during August 2011 to March 2012. The isolated 126 E. coli strains (86 from bovines and 40 from handlers) belonged to 25 different serogroups in addition to rough and untypeable strains. Using multiplex polymerase chain reaction, four EPEC strains were isolated; two each from bovines and their handlers, of which two possessed the hemolysin ( hlyA ) gene. The prevalence of EPEC was recorded as 1.66% (n=1/60) in cattle, 2.32% (n=1/43) in buffaloes, and 2.85% (n=2/70) in their handlers. Antibiogram studies with the EPEC revealed the presence of multi-drug resistant E. coli . The isolates were mostly resistant to Amikacin, Amoxicillin, Cefixime and Streptomycin, and sensitive to Chloramphenicol. This study indicates that bovines as well as their handlers in Jammu region harbor EPEC, many of which being multi-drug resistant and carrying the hemolysin gene could be of high pathogenic potential for humans. http://dx.doi.org/10.5455/javar.2014.a30

A molecular scheme for Yersinia enterocolitica patho-serotyping derived from genome-wide analysis

Yersinia enterocolitica is a food-borne, gastro-intestinal pathogen with world-wide distribution. Only 11 serotypes have been isolated from patients, with O:3, O:9, O:8 and O:5,27 being the serotypes most commonly associated with human yersiniosis. Serotype is an important characteristic of Y. enterocolitica strains, allowing differentiation for epidemiology, diagnosis and phylogeny studies. Conventional serotyping, performed by slide agglutination, is a tedious and laborious procedure whose interpretation tends to be subjective, leading to poor reproducibility. Here we present a PCR-based typing scheme for molecular identification and patho-serotyping of Y. enterocolitica. Genome-wide comparison of Y. enterocolitica sequences allowed analysis of the O-antigen gene clusters of different serotypes, uncovering their formerly unknown genomic locations, and selection of targets for serotype-specific amplification. Two multiplex PCRs and one additional PCR were designed and tested on various reference strains and isolates from different origins. Our genotypic assay proved to be highly specific for identification of Y. enterocolitica species, discrimination between virulent and non-virulent strains, distinguishing the main human-related serotypes, and typing of conventionally untypeable strains. This genotyping scheme could be applied in microbiology laboratories as an alternative or complementary method to the traditional phenotypic assays, providing data for epidemiological studies.

Phylogenetic analysis and development of an immunofluorescence assay for untypeable strains of coxsackievirus B3

In recent years, coxsackievirus B3 (CV-B3) has been determined as a dominant enterovirus serotype that may cause severe complications in patients. Since 2008 in Taiwan, some enterovirus isolates have been regarded as untypeable [by employing commercial immunofluorescence assay (IFA) kits]. In 2012, the number of isolates increased. Genetic sequence analysis further confirmed that CV-B3 was present in most of the untypeable viruses. Isolates of CV-B3 were collected for basic local alignment search tool (BLAST) analysis and for phylogenetic analyses, based on VP1 gene sequences. In addition, the Taiwan Centers for Disease Control (Taiwan CDC) developed an in-house indirect IFA using polyclonal antibodies (e.g., rabbit antisera) for diagnosis. The sensitivity and specificity were both evaluated by testing 61 reference enteroviruses and 307 local enteroviruses that were isolated between 1998 and 2010. Based on the results of the BLAST and phylogenetic analyses, five main genogroups (i.e., GI-GV) were classified and the reference strains in Taiwan in previous years were primarily clustered in the GV-A subgenogroup. However, the 15 CV-B3 isolates recently analyzed in this study were classified in four different groups: GIII, GIV, GV-A, and GV-B. Among these 15 isolates, all 10 isolates in the GV-B group were initially reported as untypeable nonpolio enteroviruses when using commercial kits. The conditions of the in-house indirect IFA were optimized by checkerboard titration, thereby resulting in a sensitivity of 100% and a specificity of 98.5%. This is the first report describing the phylogenetic relatedness of recent CV-B3 strains in Taiwan. An indirect IFA kit was developed by the Taiwan CDC for detecting CV-B3 viruses that are untypeable by commercial IFA kits.

Serogroup distribution of Shigella in Ile-Ife, southwest Nigeria

Background: Shigellosis is endemic throughout the world and Shigella spp. is among the most common pathogens responsible for bacterial diarrhoeal diseases. Death attributed to shigellosis is common in developing countries, where affected populations are immunologically compromised due to poor nutrition and background infections. Aim: To investigate the serogroup distribution of Shigella spp. recovered from clinically diagnosed cases of gastroenteritis and acute diarrhoea among children (0–5 years) in Ile-Ife, southwest Nigeria between September 2003 and September 2006. Methods: The isolates were identified and characterized biochemically and serologically. Results: Out of 102 Shigella isolates identified, 45 (44%) were S. flexneri, 26 (25%) were S. dysenteriae, 19 (19%) were S. boydii, 6 (6%) were S. sonnei and 6 (6%) were untypable strains. Conclusions: We conclude that Shigella serogroups can be considered an important aetiological agent of acute diarrhoea and mortality among children in Ile-Ife, southwest Nigeria.

Characterization of human rotaviruses circulating in Iraq in 2008: Atypical G8 and high prevalence of P[6] strains

Fecal samples from 976 children with gastroenteritis were collected and analyzed for group A rotavirus (RVA), in three different cities in Iraq between January 2008 and December 2008. RVA antigen was detected in 394 (40%) of the samples, and 98 samples were available for further genotype analyses using multiplex RT-PCR and sequence analyses for untypeable strains. The G/P-genotype combination was determined for 69 samples, and 19, 2 and 8 samples remained P-untypeable, G-untypeable and G/P-untypeable (UT), respectively. The most prevalent genotype was G2 (40%, 39/98) most often associated with P[6]. G1 was the second most common genotype (16%, 16/98) mainly associated with P[8] and P[UT]. G3, G4 and G9 were detected at a lower prevalence (3%, 11%, 3%, respectively), mainly associated with P[6]. Surprisingly, five G8P[6], and seven G12 RVA strains in combination with P[6] and P[8] were also detected for the first time in Iraq. Overall, a striking high prevalence of 47% of the analyzed samples possessed the P[6] genotype (65% of the P-typed RVA strains). Atypical genotype combinations such as G1P[4], G1P[6], G2P[8] or strains with mixed G-types were detected sporadically. The detection of unusual G8P[6] RVA strains prompted us to further analyze the NSP2, NSP3, NSP4 and NSP5 gene segments of three selected G8P[6] strains, resulting in their designation to the N2, T2, E2 and H2 genotypes, respectively. The VP7, VP4, NSP2, NSP3 and NSP5 gene segments clustered closely with common human RVA strains, whereas the NSP4 gene sequences were found to cluster with animal derived RVA strains, suggesting a potential reassortment event. The high prevalence of RVA strains with the G8, G12 and P[6] genotypes in combination with a DS-1-like genotype constellation in Iraq, needs to be monitored closely as these RVA strains might challenge the effectiveness of current RVA vaccines.

Prevalence, genetic profile of virulence determinants and multidrug resistance of Escherichia coli isolates from foods of animal origin

Aim: The aim of this study was to assess the hygienic quality of foods of animal origin. Thus samples from foods of animal origin, viz. mutton, chicken meat, milk and milk products were processed. Materials and Methods: Two hundred samples from foods of animal origin viz., mutton, chicken meat, milk and milk products were processed for isolation of Escherichia coli. The isolates were got serotyped and also subjected to detection of virulence genes viz., stx1, stx2, eaeA and hlyA by PCR.The isolates were also tested against commonly used antibiotics. Results: The prevalence of E. coli was 30% in mutton, 40% in chicken meat, 33.96% in milk and14.89% in milk products samples. All the 60 isolates of E. coli were grouped into 24 serogroups with O60 and O123dominant strains (8.33%) followed by O22 (6.66%). The PCR detected 21(10.5%) of samples possessing stx1, 14(7%) stx2, 3(1.5%) both stx1 and stx2, 16(8%), eaeA and 4(2%) EHEC-hlyA gene. However, the prevalence of Shiga toxin producing E. coli (STEC) was 20% in mutton, 30% in chicken meat, 16.98% in milk and 8.51% in milk products. Whereas the prevalence of enteropathogenic E. coli (EPEC) was 2%, in mutton, 4% in chicken meat, 7.54% in milk and 2.12% in milk products samples. The 4 isolates O60, O101, O131 and one untypeable strain possessed the EHEC-hlyA gene. 22 of 50 (44%) of isolates from meat, milk and milk products showed multidrug resistance to four or more antimicrobial comprising ten of 25 (40%) isolates from chicken meat samples and 12 of 25(48%) from milk and milk products were multidrug resistance to four or more antimicrobial. Conclusions: It is concluded that partial cooked or raw milk, meat and their products prepared under unhygienic conditions may not be directly consumed as they may be carrying the pathogenic microbes.

Prevalence of Salmonella enterica serovar 4,[5],12:i:- in England and Wales, 2010

Difficulties in accurately identifying serovar 4,[5],12:i:- as monophasic variants of Salmonella enterica serovar Typhimurium mean there is confusion in the reporting of serovars Typhimurium and 4,[5],12:i:-. To gain insight into the prevalence and diversity of these monophasic variants in England and Wales, screening for fljB, hin and the serovar 4,[5],12:i:- DT193-associated genomic island was conducted on 609 S. enterica isolates designated as definitive phage type (DT) 193, and 142 isolates serologically-defined as monophasic variants of serovar Typhimurium but belonging to phage types other than DT193. All latter 142 isolates were subtyped by multilocus variable-number tandem repeat analysis (MLVA). MLVA was also applied to 70 DT193 serologically-defined monophasic variant isolates. Results indicate that serovar 4,[5],12:i:- accounted for 108 of 209 (52%) of DT193 isolates with available serological data and 99 of 142 (70%) monophasic variant isolates belonging to other phage types. Of 609 DT193 isolates, 463 (76%) lacked fljB and hin. Moreover, genetically-related isolates of DTs 120, 191, 191a, 195, phage types U311 and U323, and reacts but does not conform (RDNC) and untypable (UT) strains were also lacking either hin and/or fljB. Of note, the serovar 4,[5],12:i:- DT193-associated genomic island was identified in not only 458 of 463 (99%) monophasic DT193 isolates, but also 25 of 139 (18%) biphasic DT193 isolates and 56 of 76 (74%) monophasic variants of other phage types. Accurate monitoring of the emergence of serovar 4,[5],12:i:- isolates is important to ascertain the public health impact of these strains; since 2012 the Health Protection Agency’s Salmonella Reference Unit has therefore begun determining full antigenic structures of all presumptive O:4 isolates in addition to routinely performing phage typing for identification of variants of serovar Typhimurium.

Community-acquired methicillin-resistant Staphylococcus aureus from skin and soft tissue infections (in a sample of Egyptian population): analysis of mec gene and staphylococcal cassette chromosome

BackgroundStaphylococcus aureus has been recognized as an important pathogen associated with inpatients and community infections. Community-acquired methicillin-resistant S. aureus (CA-MRSA) infections commonly present as skin and soft-tissue infections (SSTIs). Treatment often includes incision and drainage with or without adjunctive antibiotics. ObjectivesThis study aimed to identify CA-MRSA infections both phenotypically and genotypically, to determine their spectrum of antibiotic resistance, and to establish the best scheme for molecular distinction between hospital-acquired MRSA (HA-MRSA) and CA-MRSA by staphylococcal cassette chromosome mec (SCCmec) typing and detection of Panton Valentine leukocidin (PVL). Materials50 swabs, from skin and soft tissue of infected lesions of outpatients attending the dermatology department of the Medical School, Alexandria University, were collected. Additionally, a nasal swab was taken from every participant. MethodsCollection of swabs from the infected skin and soft tissues, followed by laboratory testing to phenotypically and genotypically identify MRSA. Also, nasal swabs were taken from every patient to identify MRSA colonization. ResultsStaphylococcus aureus strains were identified in 38 (76%) of the 50 clinical isolates. 18 (47.37%) out of the 38 S. aureus strains were resistant to oxacillin and cefoxitin discs, were penicillin binding protein 2a (PBP2a) producers, and were initially diagnosed as MRSA. All of the 18 strains were definitively diagnosed as MRSA by mecA gene detection using real time PCR, while only six (33.33%) strains were PVL positive. Using the sets of primers of Zhang et al.: nine (50%) out of the 18 CA-MRSA strains were SCCmec type V, and one (5.56%) was SCCmec type IVc. Then, using the set of primers by Oliveira et al., two (25%) out of the eight untypable MRSA strains were found to be SCCmec type IV, and six (75%) remained untypable. ConclusionsCA-MRSA must be considered when treating skin and soft tissue infections, especially in developing countries. Empirical use of agents active against CA-MRSA is warranted for patients presenting with serious SSTIs.

Human Bocaviruses Are Not Significantly Associated with Gastroenteritis: Results of Retesting Archive DNA from a Case Control Study in the UK

Gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. Despite improvements in detection methods, a significant diagnostic gap still remains. Human bocavirus (HBoV)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between HBoVs, and in particular type-2 HBoVs, and gastroenteritis has previously been made. The aim of this study was to determine the role of HBoVs in gastroenteritis, using archived DNA samples from the case-control Infectious Intestinal Disease Study (IID). DNA extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of HBoV DNA. All samples were screened in a real time PCR pan-HBoV assay, and positive samples were then tested in genotype 1 to 3-specific assays. HBoV was detected in 7.4% but no significantly different prevalence was observed between cases and controls. In the genotype-specific assays 106 of the 324 HBoV-positive samples were genotyped, with HBoV-1 predominantly found in controls whilst HBoV-2 was more frequently associated with cases of gastroenteritis (p<0.01). A significant proportion of HBoV positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. However, the distribution of the untyped HBoV strains was no different between cases and controls. In conclusion, HBoVs, including HBoV-2 do not appear to be a significant cause of gastroenteritis in the UK population.

Isolation and Molecular Genotyping of Group a Rotavirus Strains Circulating among Egyptian Infants and Children

Purpose: Group A rotaviruses are the most important cause of acute diarrhea in children throughout the world. They are the cause of more than 450,000 annual deaths. There are few data available about rotaviruses type circulating in Egypt. Genotyping by reverse transcription-PCR (RT- PCR) have been widely used. Aim: To identify rotavirus antigens and RNA among stool specimens isolated from Egyptian children and to perform molecular characterization among the detected isolates. Materials and Methods: This study comprised 450 stool specimens collected from children less than 5 years old suffering from acute diarrhea, from three different governorates in Egypt (Cairo, Sharkia and Fayoum) during May 2009 to April 2010. Results: Rotavirus was detected by RT PCR in 158 (35%) of patients stool samples. G1 was the predominant genotype detected in 87/158 cases (55%). G3 was the second most common cause and was responsible for 35 cases (22.2%). whereas G4 represented only 8.2% (13/158) of all strains. Our study identified G9 in 23 (14.5%) of positive cases. No untypable strains were detected and mixed infection between G1and G4 in 1.9% (3/158) of the samples was detected. G8, G2 were not detected among collected samples. Conclusions: These results underline the importance of continued detailed epidemiological and virological studies to identify rotavirus genotypes responsible for severe diarrhea, including characterization of the less common and or unusual strains. Focusing on the more prevalent strains circulating in the Egyptian community will add in assessing the most suitable strain candidates used for vaccine production to protect against current circulating and uncommon strains and for evaluation of cross immunogenicity among variable strains.

Detection of human G10 rotavirus strains with similarity to bovine and bovine-like equine strains from untypable samples

G10 rotaviruses, which are usually found in cattle, have also been reported in neonatal infections in recent years. During the rotavirus surveillances of children less than 4years of age between 2003 and 2006 in Kolkata, eastern India, 60 out of 1153 samples could not be typed. All 60 samples gave usual electropherotype pattern in polyacrylamide gel. Thirty-one out of these 60 G and P untypable rotavirus strains were successfully characterized during the study. Among 31 samples, G9P[4] (n=8), G12P[8] (n=8), G1P[8] (n=6), G10P[4] (n=6), and G2P[4] (n=3) genotypes were identified. In this study we report genetic analysis of the six G10 strains, which revealed close relations with Turkish (E29TR) bovine strains, as well as with bovine-like-equine strain (Erv2) from India. SimPlot of the VP7 gene segment suggested possible recombination event between the bovine and the bovine-like-equine rotaviruses in these human rotavirus infections.

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle

An atypical pestivirus (‘Hobi’-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that ‘Hobi’-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5′ untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay ( n = 2) or reacted as ‘Hobi’-like pestivirus BVDV-3 ( n = 3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true ‘Hobi’-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of ‘Hobi’-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.

Détection d’entérovirus dans les eaux usées urbaines à Yopougon, Abidjan

AimThe aim of this study was to determine prevalence of enteroviruses in raw municipal wastewater and specify serotypes circulating. MethodsAfter virus concentration by WHO recommend method, aqueous phase of 68 samples was inoculate on BGM, RD, L20B and Hep2 cells lines. Typing after titration was performed by microplate neutralization test with specific horse immune serum against EV (kit RIVM/WHO). NPEV strains were confirmed by RT-PCR test in 5′NC genomic region. Antigenic and molecular tests of Polioviruses intratypic differentiation were performed to determine vaccine or wild origin of Poliovirus strains. ResultsViral isolation obtained 43/68 (65.15 %) positives samples. Among these 35/43 (81. 39%) were EV and 8/43 (19.04 %) non EV strain. Among EVs, 34/35 (97.14 %) were NEPV and 2/35 (5.71 %) vaccinal Poliovirus strains type 2 (Sabin-like). NEPV typing gave 77 % untypeable strains by conventionally method. The average virus concentration estimated in the raw sewage is 4.28×105 PFU/L. ConclusionVarious contacts between people and these waters expose them to health risks.

Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE

Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5 (17.4%), 14 (8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil.

Gene cloning and characterization of Streptococcus intermedius fimbriae involved in saliva-mediated aggregation

Streptococcus intermedius, an oral commensal and a cause of systemic pyogenic disease, expresses fimbriae. To identify the gene(s) encoding these fimbriae, we used a serum raised against purified fimbriae to screen libaries of recombinant λ phages. The cloned gene cluster encoding S. intermedius fimbriae, ( saliva-mediated aggregation and adherence-associated fimbriae), contained 4 ORFs, i.e. a putative ribonulease (Saf1), a putative adhesin (Saf2), the main pilus subunit (Saf3) and a sortase C (SrtC). Escherichia coli strains harboring recombinant phages and plasmids carrying the saf3 gene produced a 55 kDa protein recognized by the antifimbriae serum. Saf3 contains an N-terminal signal sequence and a C-terminal cell-wall-anchoring motif LPXTG. Among strains of the Streptococcus anginosus group, only serotype g and untypable strains were found to contain the saf3 gene, to possess the fimbrial antigen and to exhibit saliva-mediated aggregation. Knockout mutants made by insertion of an erythromycin resistance gene into saf3 did not produce fimbrial structures or fimbrial antigens and did not participate in saliva-mediated aggregation. The adherent activity of mutants toward plastic wells coated with salivary agglutinin was about 65% that of the parental strain, and the reaction depended on calcium. There was no significant difference in adherence to hydroxyapatite beads pretreated with salivary agglutinin between the parental and mutant strains. These results demonstrated that Saf3 is associated with aggregation, and suggested that other molecule(s) are associated with adherence of S. intermedius.