Environmental Surveillance of Poliovirus in Sewage Water around the Introduction Period for Inactivated Polio Vaccine in Japan

Environmental virus surveillance was conducted at two independent sewage plants from urban and rural areas in the northern prefecture of the Kyushu district, Japan, to trace polioviruses (PVs) within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to January 2013. The frequency of PV isolation at the urban plant was 1.5 times higher than that at the rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the PV isolates, with the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated poliovirus vaccine (IPV) was introduced for routine immunization on 1 September 2012, replacing the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage waters declined before OPV cessation at both sites. Our study highlights the importance of environmental surveillance for the detection of the excretion of PVs from an OPV-immunized population in a highly sensitive manner, during the OPV-to-IPV transition period.     

A Double-Selective Tissue Culture System for Isolation of Wild-Type Poliovirus from Sewage Applied in a Long-Term Environmental Surveillance

We describe a simple, cost-efficient, double-selective method for isolation of wild-type poliovirus from sewage samples containing vaccine polioviruses and other enteroviruses, with a detection limit of 18 to 50 PFU per 1 to 2 liters of sewage. By this method we were able to process 1,700 sewage samples collected between 1991 and 1996, from which 10,472 plaques were isolated, 41 of them being identified as wild-type polioviruses.     

Assessment of Poliovirus Eradication in Japan: Genomic Analysis of Polioviruses Isolated from River Water and Sewage in Toyama Prefecture

Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.     

Isolation and Characterization of a Type 2 Vaccine-Derived Poliovirus from Environmental Surveillance in China, 2012

Environmental surveillance of poliovirus on sewage has been conducted in Shandong Province, China since 2008. A type 2 vaccine-derived poliovirus (VDPV) with 7 mutations in VP1 coding region was isolated from the sewage collected in the city of Jinan in December 2012. The complete genome sequencing analysis of this isolate revealed 25 nucleotide substitutions, 7 of which resulted in amino acid alteration. No evidence of recombination with other poliovirus serotypes was observed. The virus did not lose temperature sensitive phenotype at 40°C. An estimation based on the evolution rate of the P1 coding region suggested that evolution time of this strain might be 160–176 days. VP1 sequence analysis revealed that this VDPV strain is of no close relationship with other local type 2 polioviruses (n = 66) from sewage collected between May 2012 and June 2013, suggesting the lack of its circulation in the local population. The person who excreted the virus was not known and no closely related virus was isolated in local population via acute flaccid paralysis surveillance. By far this is the first report of VDPV isolated from sewage in China, and these results underscore the value of environmental surveillance in the polio surveillance system even in countries with high rates of OPV coverage.     

Incidence of Rotavirus and Circulating Genotypes in Northeast Brazil during 7 Years of National Rotavirus Vaccination

Background and Aims

Rotavirus causes severe diarrhoea and Brazil introduced the Rotarix G1P[8] vaccine in 2006. We aimed to describe changes in rotavirus incidence and diarrhoea epidemiology before and after vaccine introduction.

Methods

Design: (i) hospital-based survey of children with diarrhoea (2006–2012); (ii) diarrhea-mortality and hospitalization surveillance (1999–2012).

Setting

(i) Aracaju and (ii) state and national level.

Results

1841 children were enrolled and 231 (12.5%) had rotavirus. Rotavirus was less frequent from January-June than from July-December (9.4% versus 20.9%, p<0.01), but the seasonal variation was less defined after 2009. Very few rotavirus cases (8–3.9%) were detected in 2011, with an increase in 2012 (13–18.5%). In 2006, unvaccinated children were more likely to have rotavirus, but thereafter unvaccinated and vaccinated children had equally low incidence. Older children and those with rotavirus were more likely to have severe diarrhea episodes. The most frequent genotype from 2006 to 2010 was G2P[4]; except in 2009, when most cases were G1P[8]. Very few G2P[4] were detected from 2011 and 50% cases in 2012 were G8P[4]. Diarrhoea-hospitalizations decreased nationally from 89,934 (2003) to 53,705 (2012; 40.3% reduction) and in the state from 1729 to 748 (56.7% reduction). Diarrhoea-deaths decreased nationally from 4368 in 1999 to 697 in 2012 (84% reduction, p<0.001) and in the state from 132 to 18 (86% reduction). These changes were much larger after vaccine introduction.

Conclusions

The vaccine was associated with substantial reductions in rotavirus incidence and diarrhoea-hospitalizations and deaths. The G2P[4] genotype predominance disappeared over time and may be replaced by other heterotypic genotypes.     

Detection and prevention of protein aggregation before,during, and after purification

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.     

海口市污水处理厂活性污泥中细菌的分离鉴定及耐药性分析

为了解海口市白沙门污水处理厂活性污泥中细菌抗生素耐性情况,采用平板分离技术分离、纯化细菌,并通过BIOLOG微生物鉴定系统对筛选到的细菌进行鉴定,同时采用Kirby-Bauer纸片琼脂扩散法进行药敏试验并进行抗生素耐性分析。本研究共分离到18株细菌,分属8个属,14个种,其中G+和G-均为9株。抗生素药敏性试验结果表明,所有菌株均耐药,菌株单重耐药率、双重耐药性及多重耐药性分别为50%、38.9%、和11.1%。菌株对9种常用抗生素:头孢他啶、环丙沙星、庆大霉素、链霉素、氨苄西林、红霉素、氯霉素、四环素、卡那霉素的耐药率分别为61.1%、0%、5.6%、16.7%、50%、16.7%、11.1%、0%、5.6%。综上所述,白沙门污水处理厂活性污泥中的细菌耐药性比较严重,存在潜在的环境生态和人畜健康风险。本研究揭示了当前白沙门污水处理厂活性污泥中细菌对常见抗生素耐药的严重现状,为建议污水处理厂加强出水及污泥中抗生素耐药性及耐药基因的检测并评估其生态影响提供基础,避免出水及污泥中的抗性菌和耐药基因可能带来的风险问题。     

Molecular Characterization of Sewage-Borne Pathogens and Detection of Sewage Markers in an Urban Stream in Caracas,Venezuela

Molecular characterization of two sewage-borne pathogens identified hepatitis A virus (HAV) subgenotype IA and Giardia duodenalis assemblages A and B as predominant genotypes circulating in an urban area of Venezuela. This study reveals epidemiological features of human pathogens of worldwide distribution and the efficacy of molecular methods for accurate assessment of sewage pollution.Multiple microbial pathogens may frequently be found in surface waters that receive uncontrolled municipal sewage discharges. The range and diversity of sewage-borne pathogens in surface waters are geographically specific and strongly dependent on the burden of infectious diseases in the population, the seasonal patterns of infectious diseases in the community, and the availability of sewage treatment processing (5, 7). The metropolitan city of Caracas, the capital and largest city of Venezuela, is located in northern South America near the Caribbean coast. Water pollution is a big issue in Caracas, like in many other cities in South America, where most of the human sewage (∼97%) from overpopulated urbanized areas is discharged without any treatment into nearby rivers and coastal environments. Despite these facts, the seriousness of sewage-related health issues is not at the forefront of public concern in this country.Giardia is the protozoan parasite most frequently detected in human fecal samples submitted to diagnostic laboratories from major cities in Venezuela. The frequency of giardiasis reported in the population varies between 21% and 45% but may increase up to 75% among school age children (8). Notwithstanding, the epidemiology of giardiasis in Venezuela remains unknown, and no previous studies have documented the distribution of species and genotype assemblages associated with human infections.Hepatitis A virus (HAV), the etiological agent of hepatitis A in humans, has distinguishable epidemiological patterns of distribution and endemicity closely related to socioeconomic development (9, 12). Water- and food-borne outbreaks of HAV have been well documented worldwide (6, 19). Seroepidemiological studies conducted in selected populations in Venezuela have demonstrated high endemicity of hepatitis A infection among low socioeconomic population strata, with seroprevalences between 48% and 98% (13). Nevertheless, studies on HAV genotype circulation in major urban areas of the country are scarce, as is research on predominant exposure routes and potential transmission patterns through the environment.The analysis of nucleic acid sequences of sewage-borne pathogens may provide relevant information on predominant species and genotypes of human-pathogenic viruses and parasites circulating in specific geographical areas (11, 12, 15). The molecular approach may be of relevance for countries lacking reliable disease surveillance programs and proper understanding of the potential transmission of specific human pathogens through the environment. In this research, Giardia cysts and HAV recovered from an urban stream were characterized by multiple molecular methods along with nucleotide sequence analysis to identify predominant genotypes circulating in a major urban area of Venezuela''s capital. The strength and efficacy of multiple molecular methods for accurate assessment of human sewage pollution and risks of exposure to sewage-borne pathogens were also investigated.Dry season sampling (October through March) was conducted in a heavily polluted urban stream (>10⁶ fecal coliforms/100 ml) that flows in a southeast direction through the metropolitan city of Caracas (16). Water sample volumes of 100 ml were collected in three sterile centrifuge tubes two to four times per month. Giardia cysts were concentrated by centrifugation (100 ml at 1,500 × g for 15 min) followed by DNA extraction by the freeze-thaw method in the presence of Chelex-100 (3) for sucrose-purified cysts. Human-pathogenic assemblage occurrence was determined by nested PCR amplification and sequence analysis of the triosephosphate isomerase (tpi) gene (18). Multiple sequence alignments were performed with ClustalW (21), and phylogenetic analyses were conducted using MEGA4 software (20). The genetic diversity of Giardia isolates was inferred by the neighbor-joining method (17) using a bootstrap test of 1,000 replicates. Giardia cysts counts were obtained by fluorescence microscopy using BTF EasyStain monoclonal antibody stain (BTF Precise Microbiology, Inc.) and 4′6-diamidino-2-phenylindole (DAPI). The recovery efficiency of cysts was determined in five experiments using ColorSeed C&G spike suspensions as internal quality controls (14).HAV particles were concentrated from 35 ml by ultracentrifugation and elution with 0.25 N glycine buffer following procedures previously described (16). Viral RNA was extracted from sample concentrates with Trizol (Invitrogen, Inc., Carlsbad, CA) following the manufacturer''s instructions. General detection of HAV was based on amplification of the 5′ nontranslated region (5′ NTR), while analysis of genetic diversity involved sequencing and phylogenetic analysis of the VP1 amino terminus and the full VP1 gene (2, 10). Sequence alignment was conducted with the DNAman software 5.2.2 (Lynnon BioSoft, Quebec, Canada) followed by phylogenetic analysis.Molecular detection of sewage pollution was accomplished by PCR amplification of Bacteroidales human-specific 16S rRNA genes, Bacteroides thetaiotamicron 16S rRNA genes, and the nifH gene of Methanobrevibacter smithii using primers and PCR conditions originally described by Field et al. (4), Carson et al. (1), and Ufnar et al. (22), respectively.Giardia duodenalis tpi nucleotide sequences amplified directly from urban stream waters were included after phylogenetic analysis into two well-defined clusters of assemblages A and B. These results were supported by high bootstrap values, as indicated in Fig. ​Fig.1.1. The level of cysts recovered from these samples ranged from 10,400 to 62,000 cysts/liter; however, mean percent recoveries varied from 20% to 50%, which suggests that the urban stream may harbor and receive much higher loads of cysts.Open in a separate windowFIG. 1.Phylogenetic tree of G. duodenalis assemblages A (VPW2, VPW3, and VPW7) and B (VPW1, VPW4, VPW5, and VPW6) from urban stream samples forming two clusters in a neighbor-joining analysis of tpi nucleotide sequences. Only bootstrap values >80% are shown in the tree.Three genomic regions used for detection and characterization of HAV revealed the predominance of HAV strains belonging to subgenotype IA, the most frequent genotype associated with human disease worldwide (9). A neighbor-joining tree constructed from the alignment of nucleotide sequences from urban stream samples and sequences of HAV strains from case patients (unpublished data) was used to investigate the relationship between genotypes present in environmental and clinical samples. The comparative analysis indicated a high degree of identity (98 to 99%) between nucleotide sequences from the urban stream and the strains from sporadic HAV cases. The phylogenetic analysis grouped all of these sequences into two unique clades within subgenotype IA, strongly supported by significant bootstrap values (Fig. 2A, B, and C).Open in a separate windowFIG. 2.Phylogenetic analysis of the HAV 5′ NTR (A; 284 nucleotides [nt]), VP1 amino terminus (B; 172 nt), and complete VP1 (C; 820 nt) regions. Nucleotide sequences of HAV reference strains are designated by their GenBank accession number, including the name of the country of origin, except for Venezuelan isolates, which are shown in bold. S, isolates derived from human sporadic cases; W, urban stream isolates. Phylogenetic analysis was performed by neighbor joining, and phylogenetic distances were calculated by the Kimura two-parameter test. Bootstrap values ≥90% are shown in the trees. Letters in bold indicate the subtype.Three reliable published assays for detection of human-specific markers of fecal pollution identified and confirmed the predominant point source of water pollution. Sequence analysis of three randomly selected PCR products from each marker revealed ≥99% sequence identity with published sequences (GenBank) derived from different geographical areas, thus indicating the validity and specificity of the molecular markers as reliable indicators of human sewage pollution in Venezuela.The results of this research demonstrate that the molecular assays applied for detection and characterization of sewage-borne pathogens in surface waters may have practical applications for epidemiological investigations on distribution of predominant human-specific genotypes circulating in urban populations. Previous studies identified the most predominant waterborne gastroenteritis viruses circulating in Metropolitan Caracas (16).The molecular-based monitoring approach for rapid and precise identification of sewage-borne pathogens and sewage markers in surface waters has important implications for sewage-related health issues that require special attention in Venezuela and South America. Deficient sewerage coverage and lack of municipal wastewater treatment, commonly associated with informal settlements around densely populated urban areas, are responsible for many of the environmental degradation and public health problems that occur in these countries. The precise identification of human pathogens in the environment offers an appropriate and alternative approach for initial assessment of risks of exposure to waterborne pathogens. Current bacterial indicators of fecal pollution (fecal coliforms, Escherichia coli, and enterococci) do not allow identification of the relative sources of impacts (i.e., sewage, urban runoff, and agricultural waste) on surface waters. Thus, the molecular detection of sewage-borne pathogens and sewage markers in surface waters may be more effective than the bacterial indicator approach for forecasting pathogen distribution and for managing and reducing risks associated with inappropriate sewage disposal into natural waters in Venezuela and South America.     

A Comparison of Genetic Variation Between an Anadromous Steelhead, Oncorhynchus mykiss, Population and Seven Derived Populations Sequestered in Freshwater for 70 Years

In 1926 cannery workers from the Wakefield Fisheries Plant at Little Port Walter in Southeast Alaska captured small trout, Oncorhynchus mykiss, from a portion of Sashin Creek populated with a wild steelhead (anadromous O. mykiss) run. They planted them into Sashin Lake which had been fishless to that time and separated from the lower stream by two large waterfalls that prevented upstream migration of any fish. In 1996 we sampled adult steelhead from the lower creek and juvenile O. mykiss from an intermediate portion of the creek, Sashin Lake, and five lakes that had been stocked with fish from Sashin Lake in 1938. Tissue samples from these eight populations were compared for variation in: microsatellite DNA at 10 loci; D-loop sequences in mitochondrial DNA; and allozymes at 73 loci known to be variable in steelhead. Genetic variability was consistently less in the Sashin Lake population and all derived populations than in the source anadromous population. The cause of this reduction is unknown but it is likely that very few fish survived to reproduce from the initial transplant in 1926. Stockings of 50–85 fish into five other fishless lakes in 1938 from Sashin Lake did not result in a similar dramatic reduction in variability. We discuss potential explanations for the observed patterns of genetic diversity in relation to the maintenance of endangered anadromous O. mykiss populations in freshwater refugia.     

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed,Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine

Background

The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

Methods

qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

Results

All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

Conclusions

Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.     

Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.     

Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N⁶-(Δ²-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.

The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.

A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 × 10³ micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 × 10² micromolar) and naphthalene acetamide (3.1 × 10 micromolar) at the young fruit stage.

     

Elucidation of Echovirus 30's Origin and Transmission during the 2012 Aseptic Meningitis Outbreak in Guangdong,China, through Continuing Environmental Surveillance

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.     

Six Years after the Commercial Introduction of Bt Maize in Spain: Field Evaluation, Impact and Future Prospects

We carried out a 6-year-field evaluation to assess potential hazards of growing Compa, a transgenic Bt maize variety based on the transformation event CG 00256-176. Two categories of hazards were investigated: the potential of the target corn borer Sesamia nonagrioides to evolve resistance to Bt maize and effects on non-target organisms. In order to address the first hazard, dispersal capacity of the corn borer was measured and our results indicated that larvae move to plants other than those onto which the female oviposited - even to plants in adjacent rows - in remarkable numbers and they do so mostly at a mature age, suggesting that mixing Bt and non-Bt seeds in the same field would not be a very useful deployment strategy to delay/prevent resistance. In addition, adults move among fields to mate and males may do so for up to 400 m. Three different aspects of potential non-target effects were investigated: sub-lethal effects on the target S. nonagrioides, effects on non-target maize pests, and effects on maize-dwelling predators. Larvae collected in Bt fields at later growth stages, in which event 176 Bt maize expresses Bt toxin at sub-lethal concentrations, had longer diapause and post-diapause development than larvae collected in non-Bt fields, a feature that might lead to a certain isolation between populations in both type of fields and accelerate Bt resistance evolution. Transgenic maize did not have a negative impact on non-target pests in the field; more aphids and leafhoppers but similar numbers of cutworms and wireworms were counted in Bt versus non-Bt fields; in any case differences in damage or yield were recorded. We observed no difference in the numbers of the most relevant predators in fields containing transgenic or no transgenic maize.