Simian virus 40 (SV40) is a monkey virus that was administered to human populations by contaminated vaccines which were produced in SV40 naturally infected monkey cells.Recent molecular biology and epidemiological studies suggest that SV40 may be contagiously transmitted in humans by horizontal infection, independently from the earlier administration of SV40-contaminated vaccines.SV40 footprints in humans have been found associated at high prevalence with specific tumor types such as brain and bone tumors, mesotheliomas and lymphomas and with kidney diseases, and at lower prevalence in blood samples from healthy donors.Contrasting reports appeared in the literature on the circulation of SV40 in humans by contagious transmission and its association, as a possible etiologic cofactor, with specific human tumors. As a consequence of the conflicting results, a considerable debate has developed in the scientific community. In the present review we consider the main results obtained by different groups investigating SV40 sequences in human tumors and in blood specimens, the putative role of SV40 in the onset/progression of specific human tumors, and comment on the hypotheses arising from these data. Simian virus 40 (SV40) is a monkey virus which was accidentally administered to humans, in the years 1955-'63, through contaminated poliovirus vaccines. The subsequent findings of the transforming and oncogenity activities of the SV40 viral large T (Tag) and small t (tag) antigens, have prompted investigations into the potential of SV40 to induce cancer in humans. To date, hundreds of molecular and epidemiologic studies aimed at investigating whether SV40 infects humans, its potential mode of transmission and its putative role in human tumors, have been published.In this review we evaluate the biological, pathological and clinical evidence of SV40 in human cancers, non-malignant diseases and blood samples.Papovaviridae, an acronym proposed by Melnick and obtained by fusing the names of the three representative viruses Papilloma, Polyoma, and Vacuolating agent. More recently, SV40 has been considered a Polyomavirus, together with the human BK (BKV) and JC (JCV) Polyomaviruses. The virion is about 45 nm, an icosahedral particle, with a density of 1.34–1.35 g/cm3. The viral genome is a circular, double-stranded DNA molecule. SV40 codes, at least, for six viral proteins: two early nonstructural polypeptides, the large tumor antigen (Tag) and the small tumor antigen (tag), an agnoprotein, probably involved in the assembly of viral particles and processing of late mRNA and three capsid proteins, VP1, VP2, and VP3 [SV40 was assigned to the family of SV40 is phylogenetically closely related to the human JCV and BKV. They evidence similarity with respect to size (~5.2 Kb), genome organization and DNA sequence. The Tags of SV40, BKV and JCV strongly cross-react with the same antisera ,7, whileThe cell infection starts by the binding of the SV40 virus to a receptor on the cell membrane. This receptor has been identified as the major histocompatibility complex (MHC) . In monkDifferent studies indicate that SV40 can replicate productively in human cells, including spongioblasts, fetal neural cells, newborn kidney cells , and somTransformation of rodent and human cells by SV40 is induced by the two oncoproteins, the large Tag and the small tag, which display multiple functions. The main activity of Tag for cell transformation and tumorigenesis is to target key cellular proteins, such as the tumor suppressor p53 and pRB family proteins, inactivating their functions ,19,33-40The principal role of the small tag in transformation is to bind the catalytic (36 kDa) and regulatory (63 kDa) subunits of protein phosphatase 2A (PP2A) ,19, inacThe process of rodent cell transformation induced by SV40 typically depends on the integration of the viral DNA into the host genome where it produces a high level of expression of the major viral oncogenic proteins, large T antigen and small t antigen ,19,33-36ras oncogene, for the complete transformation of human cells, as shown in cotransfection experiments [SV40 immortalized and transformed human cells -52 are aeriments .SV40 is highly oncogenic in rodents and when inoculated subcutaneously, intracerebrally or intravenously in newborn hamsters induces soft tissue sarcomas, osteosarcomas, ependymomas and choroid plexus papillomas, and neoplasms of the hematopoietic system, such as lymphocytic leukemia, histiocytic lymphomas and rarely, B-cell lymphomas ,54-56. MSV40 natural infection in humans is considered a rare event, restricted to people living in contact with monkeys, the natural hosts of the virus, such as inhabitants of Indian villages located close to the jungle, and workers attending to monkeys in zoos and animal facilities ,62. A soEarly serologic studies reported the presence of SV40 neutralizing antibodies, at different titles, in the population that received IPV. Immune response appeared to correlate with the amount of SV40 present in the vaccine; 30% to 50% of individuals reached a significant antibody response against formalin-inactivated SV40 after three doses of the vaccine. Antibody titles persisted for a period of up to 3 years post-inoculation . AdditioTo date, the prevalence of SV40 infections in humans is not known. Recent studies, based on PCR and serological techniques, indicate that SV40 infection occurs both in children and adults. (i) SV40 DNA sequences have been detected in normal and neoplastic tissues of people either too young (1 to 30 years) or too old (60 to 85 years) to have been vaccinated with SV40-contaminated anti-polio vaccines ,33,76-81Among recent SV40 serologic investigations, some data suggest that specific SV40 antibodies are present in human sera: (i) in a collection of human sera from Morocco, 100% of the samples had antibodies to SV40 , whereasSome other recent SV40 serological studies, using virus neutralization and ELISA test, showed antibodies to SV40 in a limited number (1.3 to 15.6%) of normal human sera, suggesting a low virus circulation in the human population -104,107.The antigenic cross-reaction of SV40 with the two human polyomaviruses BK and JC has been, so far, the most difficult problem in studying the real diffusion of SV40 infection in humans. Carter et al. , using rThe site of SV40 latent infection in humans is unknown. Detection of SV40 in human kidney and urine ,85,89 poSV40 sequences were detected in kidney and cells of urine sediments from patients with focal segmental glomerulosclerosis and SV40 was isolated by co-cultivation of cells from urine sediments of such patients with CV-1 monkey cells ,85. SV40SV40 sequences have been found, mainly by PCR methods, in different human cancers including mesothelioma, osteosarcoma, and non-Hodgkin's lymphoma and some different lymphoproliferative disorders, a variety of childhood brain tumors such as ependymoma and choroid plexus tumors, as well as thyroid, pituitary and parotid gland tumors ,115-140.Some of the above mentioned studies also demonstrated the messenger RNA of SV40 Tag by RT-PCR and/or the viral oncoprotein presence by immunohistochemistry in tumor tissues ,76,129. Recently, Lopez-Rios et al.,  raised tIt has been shown that some vaccine-derived SV40 genotypes overlap with those detected in monkey and in human populations, supporting the hypothesis that contaminated vaccines may play a role in the introduction of SV40 into the human population . MoreoveAs mentioned above, SV40 sequences in human tumors were often detected at low level, less than one genome equivalent per cell , while tSuggestive data are available on the role of SV40 Tag in the pathogenesis of human mesothelioma: (i) its ability to bind in vivo p53 and RB family proteins in human mesothelioma samples ,168; (iiThe problems related to SV40 infection in the human population and to SV40 contribution to human cancer have been summarized in the recent evaluation of the "Immunization Safety Review Committee" established by the Institute of Medicine of the National Academies . The ComFM wrote the initial draft of the manuscript. AC reviewed the manuscript integrating parts of the text. VB, CP and SS gave their contributions providing comments for various sections of the text including the references. MT had the primary responsibility for rewriting, incorporating comments, answering to the reviewers and editing the final text of the revised manuscript. All authors provided comments of various drafts, participated in direction setting discussions and have read and approved the final version.


Curcumin is the major yellow pigment extracted from turmeric, a commonly-used spice in India and Southeast Asia that has broad anticarcinogenic and cancer chemopreventive potential. However, few systematic studies of curcumin's pharmacology and toxicology in humans have been performed.C3 Complex™, Sabinsa Corporation). Healthy volunteers were administered escalating doses from 500 to 12,000 mg.A dose escalation study was conducted to determine the maximum tolerated dose and safety of a single dose of standardized powder extract, uniformly milled curcumin (Seven of twenty-four subjects (30%) experienced only minimal toxicity that did not appear to be dose-related. No curcumin was detected in the serum of subjects administered 500, 1,000, 2,000, 4,000, 6,000 or 8,000 mg. Low levels of curcumin were detected in two subjects administered 10,000 or 12,000 mg.The tolerance of curcumin in high single oral doses appears to be excellent. Given that achieving systemic bioavailability of curcumin or its metabolites may not be essential for colorectal cancer chemoprevention, these findings warrant further investigation for its utility as a long-term chemopreventive agent. Curcuma longa. In India and Southeast Asia, it has long been used as a treatment for inflammation, skin wounds and tumors [Curcumin is the major yellow pigment extracted from turmeric, a commonly-used spice derived from the rhizome of the herb d tumors ,2. The cd tumors -6.C3 Complex™, Sabinsa Corporation).Curcumin appears to be safe in both large and small animal models, but few systematic studies of curcumin's pharmacology and toxicology in humans have been performed -9. MinimEligible participants were male and female volunteers, 18 years of age or older with normal organ function who had not consumed any curcumin-rich foods to their knowledge within the previous 14 days. Subjects completed a food checklist to verify that they were not consuming curcumin-rich foods. After written informed consent was obtained, three subjects were entered consecutively at dose levels from 500 to 12,000 mg. Subjects took the dose with 8 fl. oz. of water followed by a standard meal containing dietary fat .Safety was assessed for 72 hours following the curcumin dose. Toxicities were graded based on National Cancer Institute, Common Toxicity Criteria version 2.0 . The maxThe study protocol and the comprehensive written informed consent used in this study protocol were reviewed and approved by the University of Michigan Human Subject Review Board prior to the start of the study. This review panel is certified under the Federal Wide Assurance Of Protection For Human Subjects (FWA 00004969) that expires June 12, 2006. The investigators followed with annual reports, renewals, and approval throughout the duration of the study including the analysis of the data.C3 Complex™, Sabinsa Corporation). It contains a minimum 95% concentration of three curcuminoids: curcumin, bisdemethoxycurcumin, and demethoxycurcumin.Curcumin was provided in a capsule form as a standardized powder extract obtained from Alleppey finger turmeric  were enrolled in the study. The racial distribution was 18 Caucasians and 6 African-Americans. Seven adverse events occurred and all were grade 1 as summarized in Table No curcumin was detected in serum of participants administered 500, 1,000, 2,000, 4,000, 6,000 or 8,000 mg. Table C3 Complex™, Sabinsa Corporation) obtained from Alleppey finger turmeric  for up to 29 days to patients with advanced colorectal cancer and failed to detect curcumin or its metabolites in blood or urine. Serum concentrations may be dependent on the dose that is administered. Cheng et al [Low serum levels of curcumin after single dose administration is consistent with the previously reported pharmacokinetic studies in animals and humans -9,17-20.nistered  administS-transferase activity, a potential surrogate biomarker of curcumin activity, was significantly decreased in patients taking 440 mg/day despite the lack of measurable serum curcumin [Low serum levels of curcumin may be due in part to the extensive intestinal and hepatic metabolic biotransformation. Preclinical work has demonstrated that avid sulfation, glucuronidation, and reduction of curcumin occurs in the gastrointestinal tracts of rats and humans ,21-23. Acurcumin . This ficurcumin  suggest To further elucidate the cause of low serum curcumin levels after large dose consumption in the present study, the curcumin capsules were analyzed. Each capsule was found to be 75% curcumin, 23% demethoxycurcumin and 2% bisdemethoxycurcumin, which is consistent with other commercial curcuminoid products. However, the curcumin activity as measured by HPLC area count for a weighed portion of capsular content was 66% less than an equivalent weighed portion of a previously supplied, pure curcumin powder (unpublished data). Thus, curcumin bioavailability in humans and biologic activity might differ based upon the formulation used.C3 Complex™, Sabinsa Corporation) in single oral doses up to 12,000 mg appears to be excellent and warrants further investigation for its utility as a long-term chemopreventive intervention. Despite the lack of a defined maximum tolerated dose, the more important issue in chemopreventive investigation lies in determining the biologically effective dose. Future clinical investigation is required to determine such a dose. Poor bioavailability from the gut limits the systemic cancer preventive activity of curcumin and higher doses, as used in this study, may be required to overcome intestinal metabolism and achieve systemic effects. However, further investigation to determine the direct effects of curcumin on colonic mucosa are warranted to adequately assess the efficacy of curcumin as a colorectal cancer chemopreventive agent.The tolerance of curcumin (The author(s) declare that they have no competing interests.CL, MR, DB participated in concept development; study design, implementation, and coordination; and helped to draft the manuscript. DN participated in the design of the study and performed the statistical analysis. DH developed and performed the HPLC assays. CR participated in the design of the study and also developed and performed the HPLC assays. SM, JB, MB participated in the design of the study and implementation of study protocol. JC participated in concept and study design development.The pre-publication history for this paper can be accessed here:


The local environment of single nucleotide polymorphisms (SNPs) contains abundant genetic information for the study of mechanisms of mutation, genome evolution, and causes of diseases. Recent studies revealed that neighboring-nucleotide biases on SNPs were strong and the genome-wide bias patterns could be represented by a small subset of the total SNPs. It remains unsolved for the estimation of the effective SNP size, the number of SNPs that are sufficient to represent the bias patterns observed from the whole SNP data.To estimate the effective SNP size, we developed a novel statistical method, SNPKS, which considers both the statistical and biological significances. SNPKS consists of two major steps: to obtain an initial effective size by the Kolmogorov-Smirnov test (KS test) and to find an intermediate effective size by interval evaluation. The SNPKS algorithm was implemented in computer programs and applied to the real SNP data. The effective SNP size was estimated to be 38,200, 39,300, 38,000, and 38,700 in the human, chimpanzee, dog, and mouse genomes, respectively, and 39,100, 39,600, 39,200, and 42,200 in human intergenic, genic, intronic, and CpG island regions, respectively.SNPKS is the first statistical method to estimate the effective SNP size. It runs efficiently and greatly outperforms the algorithm implemented in SNPNB. The application of SNPKS to the real SNP data revealed the similar small effective SNP size  in the human, chimpanzee, dog, and mouse genomes as well as in human genomic regions. The findings suggest strong influence of genetic factors across vertebrate genomes. Single nucleotide polymorphisms (SNPs) are the most abundant genetic variation in vertebrate genomes. They have been important tools in many biological fields, including mutation mechanisms, genome evolution, disease studies, pharmacogenomics, and fine mapping -4. StronMutation at the nucleotide level does not occur randomly. Recent studies of mutational mechanisms revealed that the influence of neighboring nucleotides on SNPs was strong in the human and mouse genomes ,6,7. SpeSo far, how to efficiently estimate the effective SNP size remains unsolved. The SNPNB, an user-friendly application implemented by Java and Perl, can assist the user to evaluate and obtain a number which is close to the effective SNP size . HoweverTo estimate the effective SNP size, we designed and integrated a two-step procedure in our system Figure . In the N, we randomly generate a sub-sample of SNPs with size n0 (n0<<N). First, we calculate the frequency of each nucleotide at each neighboring site in the whole SNP data and sample SNP data, respectively. According to our previous analyses [fi,j and gi,j be the frequency of sample SNP data and whole SNP data, respectively, and Fi,j and Gi,j be cumulative frequency of sample SNPs and whole SNPs, respectively. Here i denotes one of the four nucleotides  and j denotes a neighboring site in the SNP flanking sequences (-10 to -1 at the 5' side and +1 to +10 at the 3' side). The fi,j and Fi,j of the sample SNPs are defined by:For the whole SNP data with size analyses ,7, we exgi,j and Gi,j are defined similarly except for that N, the size of the whole SNP data, instead of n0 is used. The maximum difference of cumulative frequency for each nucleotide is defined by:The Di fails to be less than 0.2%, the size of the sample set is increased by 10,000. The procedure above will run it again until the criterion of Di < 0.2% is satisfied  for the next procedure.Next, we compare the maximum difference of cumulative relative frequency of each nucleotide with the threshold value of biological significance instead of test statistic given by the KS test, because the tolerable difference in the KS test is too generous to find a reasonable sample size . Here wen, the second step is to test whether the bias patterns obtained from the sample with this size can effectively represent the bias patterns observed from the whole SNP data. This is performed by an interval evaluation using 30 different SNP subsets with size n randomly sampled from the whole SNP dataset. We choose 30 different subsets because when the sample size approaches 30, we can safely assume the distribution of the bias patterns to be normal for inference purpose by central limit theorem. For each nucleotide at each neighboring site, we calculate the bias relative to the genome sequence average  in each of the 30 sample sets, and then get its average bias  is less than its standard deviation for all nucleotides at all neighboring sites, an intermediate effective SNP size (Ne0) is found. That is, the proposed method iteratively evaluates the following difference:After getting an initial effective SNP size Bi,j - si,j, ∀ i, j     (4)|si,j is the standard deviation from the 30 bias patterns. Otherwise, we increase the sample size by 10,000 and run this step again. The procedure runs iteratively until the criterion is satisfied.where Ne0 estimates. The effective SNP size is thus the mean of these 100 estimates.The two steps above run repetitively 100 times. This leads to 100 To implement the SNPKS method, we developed computer programs in C and Perl. In the SNPKS algorithm, we need to regularly generate random numbers and then extract random SNPs from the whole SNP dataset based on the generated random numbers. This routine is computationally intensive; therefore, we wrote a computer program in C. The KS test and interval estimation were implemented in a Perl script, which calls the C program automatically. The application has been tested on both Microsoft Windows and Linux operating systems. The programs, instructions, and test data are available at the website .n0) and run the SNPKS programs iteratively. We got an initial effective size (n) 38,000. Then, we took 30 random subsets with size 38,000 and performed an interval evaluation. As shown in Figure We applied SNPKS to estimate the effective SNP size in four vertebrate genomes: human, chimpanzee, dog, and mouse. The genome-wide SNP data were retrieved from the dbSNP database of the National Center for Biotechnology Information (NCBI) (see Methods). The number of the test SNPs are shown in Table N). The comparative results suggest strong genetic influences such as CpG effects across vertebrate genomes [The effective SNP size was estimated to be 38,200 ± 2,500, 39,300 ± 2,100, 38,000 ± 2,300, and 38,700 ± 2,300 for the human, chimpanzee, dog, and mouse genomes, respectively. The 95% confidence intervals were in a narrow range in these four genomes  because the number of SNPs mapped in exons or UTRs was not sufficient. Although the numbers of SNPs and sequence compositions in the genomic regions varied greatly, their effective SNP sizes were estimated to be similar: 39,100 ± 2,300 (intergenic regions), 39,600 ± 2,200 (genes), 39,200 ± 2,200 (introns), and 42,200 ± 2,700 (CpG islands). The effective SNP size in the CpG islands is the largest. This reflects the lack of methylation and suppression of 5 islands .We compared the performance of our method versus the empirical iterative procedures in SNPNB . We testNe) is used to measure the size of an idealized population having the same effect of random sampling on gene frequency as that in the actual population. It can be estimated by Ne = θ/4 μ, where θ is the population parameter and μis the mutation rate per sequence per generation [The effective SNP size in this study is defined as the minimum number of SNPs that are sufficient to represent the bias patterns observed from the whole SNP data. It measures the sequence context patterns observed in the SNPs. To our knowledge, this term has not been used in any other report except for our previous study . This teneration . One exaneration .It is important to know how many SNPs are sufficient to represent the bias patterns observed from the whole SNP data. First, this evaluates whether the observed patterns are representative or random in the genome ,19. The P = 0.83). While this may suggest the strong influence of genetic factors in these genomes, further investigations on these factors and SNP ascertainment biases are warranted. Note that, because the size was increased by 10,000 each time in the iterative procedure in SNPKS, it is unlikely the method led to the similar effective sizes. Further, the effective sizes of the SNPs among the human genomic regions were overall slightly higher than the genome-wide whole SNPs . If we apply the Bonferroni corrections for 80 multiple comparison tests, we have the significance level α/80 for each test . That men Figure . HoweverWe proposed an integrated statistical method (SNPKS) to estimate the effective SNP size. SNPKS consists of two major steps: evaluation of an initial effective size and iterative tests of the size by interval evaluation. SNPKS considers both the biological significance and the statistical significance. SNPKS is the first method to estimate the effective size based on statistical tests and greatly outperforms SNPNB. The application of SNPKS to real SNP data in the human, chimpanzee, dog, and mouse genomes revealed the similar small effective SNP size  in these four genomes and in human genomic regions, suggesting strong influence of genetic factors across vertebrate genomes.We downloaded SNPs in four vertebrate genomes  from the dbSNP database . We retrWe selected only those SNPs that were biallelic, mapped in the non-repetitive sequences, and at least 20 nucleotides long at each side of flanking sequences. A SNP was assigned in the non-repetitive sequences if the 20 nucleotides at each side of the SNP did not overlap any repeats. A total of 5,200,425 (human), 1,470,501 (chimpanzee), 2,690,084 (dog), 7,832,159 (mouse Build 126), 376,146 (mouse Build 123), 861,498 (HapMap phase I), and 2,435,362 (HapMap phase II) SNPs were extracted after this data process (Table CpG/ExpCpG ≥ 0.65, and length ≥ 500 bp to screen CpG islands in the human genome sequences. The criteria above can effectively exclude the universal Alu repeats, which typically have a sequence length of 300 bp, GC content of 53%, and ObsCpG/ExpCpG ratio of 0.62 [We next identified SNPs in human genomic regions using the procedures described in Jiang and Zhao . First,  of 0.62 ,34. We iDS participated in the data preparation and the method development, carried out computational work, and helped to draft the manuscript. CJ participated in the data preparation and analysis. ZZ conceived of the study, participated in its design, method development, and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Bias comparison using genome-wide dog SNPs. Supplementary Table S1 – Bias comparison using genome-wide dog SNPs.Click here for file


This study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disase virus (vvIBDV) in SPF chickens.Two groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR) were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX) RT-PCR.In the tripple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups.The results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert. Gumboro disease, subsequently named infectious bursal disease (IBD) is a disease in young chickens caused by infectious bursal disease virus (IBDV), a double stranded, bi-segmented RNA virus . Two serSalmonella [E. tenella [Infection with virulent IBDV in three to six weeks old chickens causes high morbidity and mortality, followed by immunosuppression in surviving chickens . Exposurlmonella , infectilmonella , Newcastlmonella  and E. t tenella ,14. HoweEimeria species are regarded as ubiquitous parasites in most poultry environments, colonizing chicken guts after oral uptake of sporulated oocysts. Coccidial infections are traditionally controlled by coccidiostats in the feed. However, in Denmark whole wheat is gradually added to the feed, and from the age of three weeks and until processing, 25–30% of the broiler feed may be substituted by wheat. The concentration of coccidiostats decreases proportionally, increasing the risk of subclinical coccidiosis. E. tenella mainly replicates in the epithelium of the cecae, but developing stages of E. tenella have been found in the bursa of Fabricius [et al. [Problems due to IBDV outbreaks have been reported from Denmark  and seveabricius , also inabricius . Developabricius , while pabricius , also deabricius . Indeed, [et al.  have repParameters investigated and compared included clinical signs, pathological lesions in the intestines and in the bursa of Fabricius, seroconversion, and presence of viral RNA in lymphoid tissues and bursal swab samples.SPF eggs from Lohmann Tierzucht  were hatched under laboratory conditions, and chickens were reared as described previously . The metVirus strains used included the commercially available IBD vaccine strain D78 and the virulent strain DK01, previously described .E. tenella oocysts from a Swedish field outbreak were kindly provided by Dr. P. Thebo, S.V.A., Uppsala. The oocysts were sporulated and kept in 2% potassium dichromate solution at 12°C [E. tenella oocysts per ml and approximately 10 E. acervulina oocysts per ml, as estimated by counting in a McMaster chamber under a microscope. The dose of sporulated oocysts was adjusted to infect the birds without causing mortality [E. tenella oocysts were given orally in the following doses, 0, 150, 1500 and 15 000. The chickens were euthanised seven days after inoculation, and autopsy was performed immediately to evaluate the degree of coccidial infection. Lesion score results, evaluated as described under detection of coccidia, were as follows: 0,0,0; 0,0,0; 3,2,2 and 3,3,3. From these results it was decided to inoculate three experimental groups with 1500 sporulated oocysts each, in order to mimic subclinical coccidiosis. at 12°C . The solortality . The optJohnson & Reid [E. tenella, 0 = no gross lesions, 1 = few scattered petechiae on the cecal wall, normal cecal wall and contents, 2 = more numerous petechiae, cecal wall somewhat thickened, blood present in cecal contents, 3 = coalescent petechiae, cecal walls greatly thickened, much blood and fibrin cloths in cecal contents, 4 = Cecal walls greatly swollen and thickened, distended with blood or caseous clots. Concerning E. acervulina, 0 = no gross lesions, 1 = few elongated white patches in the duodenal wall in a ladder-like aspect, normal intestinal wall and contents, 2 = numerous white patches in the duodenal wall, normal intestinal wall and contents, 3 = coalescent lesions in the entire duodenum, thickened intestinal wall, contents watery and slimy, 4 = completely coalescent lesions, greyish mucosa, greatly thickened intestinal wall, mucoid contents.The intestinal tract was removed immediately after death and examined macroscopically under strong light. Lesion scores were evaluated according to n & Reid . ConcernIn addition, a smear was prepared from duodenum, jejunum and cecum in order to confirm the presence of coccidia by examination directly under light microscope.Bursa tissues were fixed in 4% formaldehyde overnight, embedded in paraffin wax and cut in 2 μ sections, mounted on Super Frost Plus slides and stained with hematoxylin and eosin (HE) .Lesions observed in bursa tissues were quantitatively transformed. Bursa samples showing no lesions were assigned a value of 0, lesions involving between one and 25% of the follicles was assigned a value of 1, 26 to 50% was assigned a value of 2, 51 to 75% was assigned a value of 3, and 76 to 100% affected follicles was assigned a value of 4.Serum samples were stored at -20°C until analysed. The Infectious Bursal disease Antibody Test Kit 113 from BioChek B.V.  was used as recommended by the manufacturer. Microsoft Exel was used for calculating the IBDV ELISA titres according to instructions from the manufacturer. Microsoft Excel was also used for calculation of average titres within groups including standard deviations, graphically illustrated.RNA extraction and the RT-PCRs were performed as previously described ,26.Four groups 1–4), including 23, 24, 23 and 23 chickens respectively were vaccinated as recommended  with strain D78 at the age of 21 days. At the age of 24 days, chickens in groups 1, 3 and 4 were inoculated orally with 1500 sporulated oocysts each. Feed was withdrawn four hours before inoculation to facilitate the flow of the oocysts into the gut. Then, 0.225 ml of the solution of sporulated oocysts was diluted in 5.775 ml tap water, and 0.2 ml of this mixture was inoculated into each chicken orally. Groups 2, 3 and 4 were subsequently challenged with vvIBDV at the age of 28 days  on day 31, seven days after inoculation, and at the same time numerous unsporulated oocysts, corresponding in morphology and size to E. tenella were not observed in any tissue samples from the bursa of Fabricius.Developmental forms of Gross pathological changes due to IBDV were only related to the bursa of Fabricius. Randomly occurring slight enlargements of the bursa were observed on day 28, one week after vaccination. Microscopic lesions were observed in all bursa tissue samples except one from a 38-days old chicken from group 5. The vaccine strain initially caused lymphocyte depletion, interstitial oedema and folding of follicular epithelium. After challenge with DK01, lymphocyte depletion became more pronounced, as vacuoles and necrotic cells were observed in the follicular medulla, and the structure of the follicles was dissolved. Bursa lesions observed in groups 3 and 4 included a larger part of the bursa tissue than the lesions observed in groups 1 and 2 on days 28 or 29. The lesion score decreased with time after 30 days in group 1, while the lesion scores in groups 2, 3 and 4 either remained high or increased after challenge with field virus on day 28 .In group 1, viral RNA identified as D78 was found in bursa tissues only, 96% of these samples being positive Table . In grouTwo days after inoculation with bursa extract, the eleven bursa-inoculated chickens were clinically ill and consequently euthanized. Autopsy revealed swollen bursa of Fabricius and petechial muscular bleedings. DPX RT-PCR using tissue from the bursa of Fabricius detected both D78 and DK01 in all bursa samples (data not shown).E. tenella as previously reported [Although clinical symptoms were not observed, lesion scores and microscopy documented subclinical coccidiosis in groups 1, 3 and 4 two days after the chickens in groups 3 and 4 were challenged with field virus. From the same day, unprotected chickens would be expected to show clinical IBD symptoms . The prereported  could beBursa lesion scores were more severe for groups 3 and 4 than for group 2, except for group 4 on day 30 and both groups on days 38 and 44. The presence of coccidia might have aggravated the development of lesions in the bursa caused by IBDV. As the number of samples in each age group was too small for reliable statistical evaluation of results, further investigations are needed for documentation of a potential influence of coccidia on bursa lesions caused by IBDV.Serology was used only in order to confirm, that all chickens had seroconverted. Titre values were not further interpreted, as this would require investigations into the technical performance of the ELISA-kit used, falling outside the scope of this work .Zhang et al. [p.i. We speculated that this could indicate that vaccine strains generalize and replicate continuously in various lymphoid tissues, but at a lower level than in the bursa, and thus mostly undetectable by our current methods, until replication was enhanced in groups 3 and 4. As enhanced replication in the spleen and thymus was not recorded in groups 1 and 2, the present reaction seemed to depend on a concurrent stimulation of both coccidia and vvIBDV. Rautenschlein et al. [D78 was detected in spleen and thymus tissues from groups 3 and 4 but not from group 2, indicating that the presence of coccidia may have influenced the distribution of vaccine strain RNA. However, this theory was impaired by the lack of D78 in the spleen and thymus tissues from group 1. g et al.  detectedn et al.  suggesten et al. . The sigIn conclusion, coccidia did not seem to affect IBDV vaccination in chickens negatively. On the contrary, our results suggested an additive effect of concurrent stimulation of the immune system by subclinical coccidiosis and vvIBDV, enhancing the replication and distribution of the vaccine strain in chicken lymphoid tissues. Assuming that replication of and presence of vaccine virus in extra-bursal lymphoid tissues mediates improved protection, our experiment indicated that coccidia contributed to an improved immune response following IBDV vaccination. The perspectives of these conclusions might be a possibility of benefiting from an enhancing immunological effect of concurrent, controlled viral and parasitic infections. Further research into immunological consequences of complex infections in chickens is highly relevant.The author(s) declare that they have no competing interests.SK designed and carried out the experiments and drafted the manuscriptKJH supervised and adjusted laboratory processesMB conceived of the study and participated in drafting the manuscriptAll authors read and approved the final manuscript


CLDN) genes encode a family of proteins important in tight junction formation and function. Recently, it has become apparent that CLDN gene expression is frequently altered in several human cancers. However, the exact patterns of CLDN expression in various cancers is unknown, as only a limited number of CLDN genes have been investigated in a few tumors.The claudin (CLDN genes from Genbank and we used the large public SAGE database to ascertain the gene expression of all 21 CLDN in 266 normal and neoplastic tissues. Using real-time RT-PCR, we also surveyed a subset of 13 CLDN genes in 24 normal and 24 neoplastic tissues.We identified all the human in silico analysis and RT-PCR data, we find that most claudin genes appear decreased in cancer, while CLDN3, CLDN4, and CLDN7 are elevated in several malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes, ovary, stomach, colon, breast, uterus, and the prostate. Interestingly, CLDN5 is highly expressed in vascular endothelial cells, providing a possible target for antiangiogenic therapy. CLDN18 might represent a biomarker for gastric cancer.We show that claudins represent a family of highly related proteins, with claudin-16, and -23 being the most different from the others. From CLDN gene expression patterns and identifies new ones, which may have applications in the detection, prognosis and therapy of several human cancers. In particular we identify several malignancies that express CLDN3 and CLDN4. These cancers may represent ideal candidates for a novel therapy being developed based on CPE, a toxin that specifically binds claudin-3 and claudin-4.Our study confirms previously known  CLDN1) [CLDN14) [CLDN16)[The claudin family consists of approximately 23 proteins that are essential for the formation of tight junctions (TJs) in epithelial and endothelial cells . TJs havCLDN3, and CLDN4 have been found frequently up-regulated in ovarian, breast, prostate and pancreatic tumors [CLDN7 has been found downregulated in breast and head and neck cancer, but elevated in stomach cancer [CLDN1 is typically downregulated in various cancers, but has also been reported to be elevated. The picture that emerges suggests that claudin expression is altered in several human tumors. Specifically, CLDN1,3,4,5,7,10,16 have been found altered in various cancers [Recent gene expression profiling analyses have shown that claudin gene expression is frequently altered in various cancers . For c tumors -11. CLDNh cancer ,13. CLDN cancers ,14,15.Clostridium perfringens enterotoxin (CPE) as a novel chemotherapeutic compound. CPE is a natural ligand for claudin-3 and -4 proteins, and binding of the toxin to these claudins leads to a rapid cytolysis of the cells [Regardless of their exact functions in cancer cells, claudin protein expression may have significant clinical relevance ,6. For ehe cells . Recent he cells ,9,11,20.Unfortunately, the exact patterns of expression of the various claudins in different cancers and normal tissues are not well known. To date, only a few of the claudin proteins have been investigated in a relatively limited number of cancers. In this report, we use the vast amount of data present in the public SAGE database to create a claudin gene expression profile of all the known claudin genes, in a large number of tissues. We then survey a subset of these claudin genes using real-time RT-PCR in a panel of normal and neoplastic tissues. Our study confirms previous claudin gene expression patterns and identifies new ones, which may potentially be of clinical use for various cancers.21 human claudin genes and corresponding proteins sequences were identified and downloaded from GenBank. The ClustalW software (with the Blosum62 matrix) was used to produce a multiple sequence alignment of all these human claudin protein sequences and the Jalview software was then used to visualize the results .GAPDH or ACTB yielded a total of 266 SAGE libraries with at least some level of expression of these control genes. These libraries were then examined for the expression of all 21 human CLDN genes that we identified. SAGE data for both normal and cancerous tissues was exported to an excel spreadsheet, and expression levels converted to tags per 200,000 . The dChip software  program was then used to visualize this data, assigning darker shades of red to higher number of tags.Mining of the SAGE Genie database  for librCLDN genes  and the control GAPDH were designed to cross intron-exon boundaries to distinguish PCR products generated from genomic versus cDNA template. For CLDN genes lacking introns, real-time RT-PCR was performed by the polyA cDNA-specific RT-PCR method [A total of 48 cDNA preparations from various normal and neoplastic tissues 24 each) were purchased from Biochains . The GeneAmp 7300 Sequence Detection System (PE Applied Biosystems) was used for detecting RT-PCR products in real-time with the SYBR Green I assay, as previously described [ each werR method . The priEach PCR reaction was optimized to ensure that a single band of the appropriate size was amplified and that no bands corresponding to genomic DNA amplification or primer-dimer pairs were present. The PCR cycling conditions were performed for all samples as follows: 50°C, 2 minutes for AmpErase UNG incubation, 95°C, 10 minutes for AmpliTaq Gold activation, and 40 cycles for the melting  and annealing/extension (60°C for 1 minute) steps. PCR reactions for each template were done in duplicate in 96-well plates.T method  was used to determine relative quantitation of gene expression for each CLDN gene compared to the GAPDH control. First, the CT values from GAPDH reactions were averaged for each duplicate. Next, the relative difference between GAPDH and each duplicate was calculated as previously described [CLDN genes was performed with distances based on 1-rank correlation and the centroid linkage method.The comparative Cescribed . The finAs a starting point for our analyses, we identified 21 different human claudin proteins in the GenBank database. Alignment of these 21 sequences using ClustalW shows that most of the claudin proteins are extremely similar, especially in the membrane-spanning regions Figure . Notablein silico analysis of gene expression and comparison of transcript levels in a large number of normal and diseased tissues [CLDN1,2,3,4,5,7,11,12, and 15 are expressed in a large number of different tissues. In contrast, other claudins, such as CLDN14,16,17,20, and 22 have much more restricted expression patterns. CLDN17, for example, was found expressed in only one SAGE library , at low levels. CLDN20 was only found in 3 cancer libraries total . Similarly, CLDN22 was only found in 2 breast cancer libraries and one brain astrocytoma library. On the other hand, CLDN3,4, and 7 were highly expressed in most normal epithelial cells as well as their corresponding neoplasias. Further analysis suggested that these claudins are frequently elevated in cancer. For example, CLDN3 was elevated in tumors of the lung, prostate, breast, kidney, and ovary compared to their normal counterparts. Similarly, CLDN4 was elevated in tumors of the lung, breast, stomach, pancreas, and ovary. CLDN7 was elevated in cancers of the thyroid, lung, stomach, pancreas, liver, kidney, and ovary. Interestingly, CLDN6 was frequently expressed in embryonic stem cells (ESCs) but generally not in other tissues. CLDN7 was the only other CLDN expressed at significant level in ESCs. CLDN18 expression seemed to be mostly restricted to the stomach and the lungs. Vascular endothelial cells expressed CLDN5 at high levels, suggesting a new target for antiangiogenic therapy. Brain had distinctive claudin expression profiles, with CLDN3,4 and 7, expressed at low levels but CLDN2 and CLDN5 very highly expressed. This pattern was opposite to what we observed in epithelial cells. CLDN12 was the most widely expressed gene and appeared expressed constitutively in most tissues.Because serial analysis of gene expression (SAGE) measures absolute levels of transcripts, cross-comparison of SAGE data is possible across experiments and laboratories . The SAG tissues . Using tin silico results obtained with the SAGE Genie database, we performed real-time RT-PCR analysis on a subset of CLDN genes  to survey gene expression in several tissues. Gene-specific primers for CLDN1,2,3,4,5,7,8,9,10,11,12,16,18 were designed and optimized. These CLDN genes were chosen because they were expressed at detectable levels in several tissues and represented a wide variety of different expression patterns as suggested by the SAGE database analysis. A total of 24 normal tissues and 24 tumors were surveyed for CLDN expression . Clustering of CDLN genes showed a tight association of CLDN3,4,7 in terms of their expression patterns, suggesting a coordinate regulation of these genes. The CLDN3,4,7 cluster was found expressed at high levels in normal pancreas, salivary gland, kidney, adrenal gland, small intestine, colon, and thyroid. Examination of the tumor samples revealed that the diversity of CLDN expression was decreased in these samples  is striking. Additional studies with several tumors, including various subtypes, grades, stages, and other clinical parameters will be necessary.In order to validate and extend the s Figure . For exas Figure . Except CLDN genes we have identified in GenBank. We find that, while some CLDN genes are ubiquitously expressed, the majority of these genes exhibit a very restricted expression pattern. CLDN14,16,17,19,20, and 22, for example, are found in only a few rare libraries. Others such as CLDN3,4,5,7,11, and 12 are much more widely expressed. Our analysis allows for the identification of general expression patterns, such as the high expression of CLDN3,4 and 7 in epithelial tissues, and lower expression in other tissues, such as the brain.Alterations in the expression levels of tight junction proteins, especially claudins, continue to be reported in several cancers. However, an overall view of claudin gene expression in normal and cancer tissues has been lacking. In this report, we first use the large public SAGE database to investigate claudin expression of the 21 human CLDN18, while other tissues do not express this gene. Interestingly, a recent study shows that claudin-18 is highly expressed in normal gastric cells and that this high expression is retained in approximately half the gastric tumors [CLDN5 is not generally expressed in epithelial tissues but is expressed at high levels in all vascular endothelial cell libraries analyzed. Although also expressed in the brain, CLDN5 may represent a target for antiangiogenic therapy, especially if using compounds that cannot cross the blood-brain barrier.Our data also reveal claudin expression patterns that were not previously known and that may have clinical implications. According to our data, gastric cells  express high levels of c tumors . BecauseCLDN gene expression, but rather a survey of expression in a large number of different human tissues. Follow-up studies on multiple samples for these different malignancies will be necessary to clearly establish the extent and levels of expression of claudins in these tissues. However, it is important to note that the patterns of gene expression obtained by real-time RT-PCR . In addition, we also observe high correspondence in the two approaches when examining CLDN5 expression, which appears to be especially high in normal brain and brain cancer. Interestingly, when clustering our RT-PCR data for gene expression patterns, we find CLDN3,4,7 are very similar in their expression, suggesting coordinate regulation. The fact that the CLDN3,4,7 cluster is present in both normal and tumors suggests that the mechanisms that lead to the coordinated expression of claudins in normal cells is conserved in tumor cells, although it may be inappropriately activated in cancer. It will certainly be interesting to elucidate the mechanisms that lead to the inappropriate activation of these genes.Our RT-PCR experiments provide a more quantitative look at claudin gene expression in several normal and neoplastic tissues. It is important to note that these RT-PCR investigations do not represent an exhaustive study of R Figure  closely e Figure . For exain silico and RT-PCR results are consistent with numerous previous reports showing that CLDN3 and CLDN4 are overexpressed in breast [CLDN4 in pancreatic cancer is also in agreement with previous reports [CLDN3, and CLDN4 are expressed in several normal tissues  declare that they have no competing interests.KJH: Performed RT-PCR experiments, multiple alignments, and drafted a first draft of the manuscript; RA: Designed and tested some of the primers for the RT-PCR experiments; PJM: Designed and coordinated the study, interpreted the data, and drafted the manuscript.The pre-publication history for this paper can be accessed here:


It has been suggested throughout the past fifty years that serum uric acid concentrations can help predict the future risk of coronary heart disease (CHD), but the epidemiological evidence is uncertain.We report a “nested” case-control comparison within a prospective study in Reykjavik, Iceland, using baseline values of serum uric acid in 2,456 incident CHD cases and in 3,962 age- and sex-matched controls, plus paired serum uric acid measurements taken at baseline and, on average, 12 y later in 379 participants. In addition, we conducted a meta-analysis of 15 other prospective studies in eight countries conducted in essentially general populations. Compared with individuals in the bottom third of baseline measurements of serum uric acid in the Reykjavik study, those in the top third had an age- and sex-adjusted odds ratio for CHD of 1.39  which fell to 1.12  after adjustment for smoking and other established risk factors. Overall, in a combined analysis of 9,458 cases and 155,084 controls in all 16 relevant prospective studies, the odds ratio was 1.13 , but it was only 1.02  in the eight studies with more complete adjustment for possible confounders.Measurement of serum uric acid levels is unlikely to enhance usefully the prediction of CHD, and this factor is unlikely to be a major determinant of the disease in general populations. The largest ever prospective analysis and meta-analysis of uric acid in coronary heart disease finds no evidence that uric acid is useful in predicting coronary heart disease Numerous genetic and environmental factors have been associated with uric acid , and serTo help address these uncertainties, we report a prospective study with more CHD cases than any previous report on serum uric acid, involving 2,459 incident cases of nonfatal myocardial infarction (MI) and CHD death, and 3,969 controls from within a prospective observational study of about 19,000 middle-aged Icelanders without a previous history of MI. To help put these results in context, we also report a meta-analysis of 15 previously published prospective studies of serum uric acid, involving a total of an additional 7,002 incident CHD cases and an additional 151,122 controls, including supplementary information obtained by correspondence from investigators to help assess in more detail the impact of possible confounders. The present analyses have been restricted to prospective cohorts sampled from essentially general populations  to reduce any distorting effects of preexisting disease on serum uric acid levels.The Reykjavik Study, initiated in 1967 as a prospective study of cardiovascular disease, has been described in detail previously [Serum uric acid levels were measured with a Technicon autoanalyzer . The meaCase-control comparisons were made by unmatched stratified logistic regression fitted by unconditional maximum likelihood. Analysis of serum uric acid values was previously specified to be by sex-specific thirds of values in the controls . Adjustment was made for age, sex, smoking status , daily cigarette consumption, blood pressure, body mass index, fasting concentrations of total cholesterol and triglycerides, and various markers of socioeconomic status related to occupation, education, home ownership, and type of accommodation. We assessed variation in the strength of association according to pre-specified sub-groups, using likelihood ratio tests for interaction after adjusting for these factors, with 99% confidence intervals (CIs) used in these exploratory analyses.For the meta-analysis, studies of serum uric acid and CHD published before May 2003 with greater than a year's follow-up conducted in essentially general populations  were sought by computer-based searches, scanning the reference lists of all relevant studies and review articles, hand-searching of relevant journals, and correspondence with authors of studies. Computer searches using Medline, PubMed, Web of Science, and Embase databases used keywords relating to uric acid in combination with CHD . Relevant endpoints included nonfatal MI  and CHD death .The following factors were abstracted from each study: numbers of cases and controls, mean age of cases and percentage of males, mean duration of follow-up, assay type, and those used for adjustment in multivariable assessments. Five studies were excluded because they reported insufficient data or only unadjusted risk ratios ,11,12,13p < 0.0001 for each), although most of these associations weakened after adjustment for other vascular risk factors  y. There were highly significant differences between cases and controls with respect to established vascular risk factors such as smoking, body mass index, blood pressure, and serum lipid concentrations . Serum u factors . In 379 21 = 18.4) in males in the top third compared with those in the bottom third of baseline serum uric acid levels  after adjustment for smoking, other established risk factors, and indicators of socioeconomic status (21 = 9.1) in females in the top third compared with those in the bottom third of baseline serum uric acid levels , and this fell to 1.12  after adjustment for smoking, other established risk factors, and indicators of socioeconomic status. In a combined analysis of males and females, the odds ratio for CHD was 1.39  and this fell to 1.12  after adjustment. In analyses restricted to the 2,083 cases without evidence of CHD at baseline, the adjusted odds ratios fell further to 1.08  in males and 1.00  in females .The odds ratio for CHD was 1.39 , and thc status . The odd females , but the females  legend. 21 = 1.1; p = 0.3). In a subsidiary analysis of seven studies [22 = 38.1, p = 0.02), but only some of this was explained by study characteristics such as sample size (χ22 = 11.1), geographical location (χ22 = 1.0), sampling framework (χ21 = 0.5), degree of adjustment for possible confounders (χ22 = 10.0), duration of follow-up (χ21 = 0.1), and assay type (χ23 = 4.2) .In aggregate, 16 prospective reports ,26,27,28 studies ,24,25,273 = 4.2) . In a raThe present report provides prospective evidence from the largest study so far of serum uric acid and CHD—plus a meta-analysis of 15 previous relevant studies—involving a total of more than 9,000 incident cases and more than 150,000 controls. The overall findings suggest that individuals with baseline serum uric acid values in the top third of the population have about a 10% greater risk of CHD over the subsequent decade than those in the bottom third . It is likely, however, that this modest association has been exaggerated by the preferential publication of striking findings in smaller studies (“publication bias”), or by residual confounding by established risk factors, or both. For example, the observation of weaker associations in studies with more comprehensive adjustment for possible confounders lessens the likelihood that any association between serum uric acid and CHD is independent from possible confounders; the odds ratio was only 1.02 , which is not significant, in the eight studies with the most complete reported adjustment for possible confounders . The preThe main implication of these data is to refute suggestions made throughout the past half-century that measurement of serum uric acid can importantly enhance the prediction of CHD in general populations. These data do not directly address the question of whether or not serum uric acid may be involved in the causation of CHD through a number of potentially relevant vascular effects , butTable S1(67 KB DOC).Click here for additional data file.Defining which risk factors are important for disease is useful for clinicians and patients not only because the presence of risk factors allows the prediction of who is more likely to get a disease, but also because they provide some insight into the underlying causes of disease. One such suspected risk factor for coronary heart disease is the level of uric acid in the blood. The debate over whether uric acid is useful for predicting heart disease has been going on for over fifty years. Most evidence for risk factors comes from studies of populations, in which it can be hard to tease out the effects of many different factors; often studies come to different conclusions. One way of finding out which results are reliable is to pool the results of many studies.They looked at the uric acid levels of around 2,500 people with coronary heart disease and almost 4,000 controls measured at the start of a large study in Iceland, and then investigated whether there was a relation between levels of uric acid and development of heart disease. After adjusting for all the other factors that could affect the chance of heart disease, they found that uric acid did not predict heart disease. They then combined these results with those from 15 other studies, and confirmed the findings.After fifty years, it now seems clear that measurement of uric acid does not help to predict heart disease. It may still be involved in triggering heart disease, but any effect must be subtle.http://www.nhlbi.nih.gov/health/public/heart/index.htm#amiThe National Heart Lung and Blood Institute has many pages of information on heart disease:


Mitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although complete mitochondrial genome sequences have been reported for more than 600 animals, the taxonomic sampling is highly biased toward vertebrates and arthropods, leaving much of the diversity yet uncharacterized.Nautilus macromphalus, a cephalopod mollusk, is 16,258 nts in length and 59.5% A+T, both values that are typical of animal mitochondrial genomes. It contains the 37 genes that are almost universally found in animal mtDNAs, with 15 on one DNA strand and 22 on the other. The arrangement of these genes can be derived from that of the distantly related Katharina tunicata (Mollusca: Polyplacophora) by a switch in position of two large blocks of genes and transpositions of four tRNA genes. There is strong skew in the distribution of nucleotides between the two strands, and analysis of this yields insight into modes of transcription and replication. There is an unusual number of non-coding regions and their function, if any, is not known; however, several of these demark abrupt shifts in nucleotide skew, and there are several identical sequence elements at these junctions, suggesting that they may play roles in transcription and/or replication. One of the non-coding regions contains multiple repeats of a tRNA-like sequence. Some of the tRNA genes appear to overlap on the same strand, but this could be resolved if the polycistron were cleaved at the beginning of the downstream gene, followed by polyadenylation of the product of the upstream gene to form a fully paired structure.The mitochondrial genome of the bellybutton nautilus, Nautilus macromphalus mtDNA contains an expected gene content that has experienced few rearrangements since the evolutionary split between cephalopods and polyplacophorans. It contains an unusual number of non-coding regions, especially considering that these otherwise often are generated by the same processes that produce gene rearrangements. The skew in nucleotide composition between the two strands is strong and associated with the direction of transcription in various parts of the genomes, but a comparison with K. tunicata implies that mutational bias during replication also plays a role. This appears to be yet another case where polyadenylation of mitochondrial tRNAs restores what would otherwise be an incomplete structure. Animal mitochondrial DNA (mtDNA) is nearly always a closed circular molecule and, with a few exceptions e.g. -4], cont, cont4]]N. macromphalus .Little study has been done to date on mollusk mtDNAs compared to those of vertebrates or arthropods , but it Nautiloids were once abundant and diverse in the Paleozoic seas, but only a handful of species remain. They are part of the molluscan class Cephalopoda, which otherwise contains octopi, squid, and cuttlefish. They are the earliest diverging lineage of this group and are often considered to be "living fossils" since living forms seem to have changed little from their ancient ancestors. They live in spiral-shaped shells which are filled with gas to control buoyancy and they move about by squirting jets of water. They are carnivorous, using their many grooved tentacles to grasp prey and pass it to their mouth, where a beak-like jaw tears it and passes it to the shredding radula. They live throughout the Southwest Pacific Ocean, at depths as great as 610 meters, and traverse a great range, as shallow as 90 meters, apparently in search of prey.Katharina tunicata) [Octopus vulgaris [Loligo bleekeri [Todarodes pacificus [O. ocellatus, Sepioteuthis lessoniana, Watasenia scintillans, and Sepia officinalis [N. macromphalus mtDNA with those of some other mollusks are presented here.Complete mtDNA sequences have been determined for 23 mollusks see , includiunicata)  of a basvulgaris , Loligo bleekeri , Todarodacificus ,31, O. oNautilus macromphalus  mitochondrial genome is 16,258 bp in length (GenBank accession number DQ472026) and contains the set of 37 genes most commonly found for animal mtDNAs [trnQ and trnT.Complete mtDNA sequences have been determined 23 mollusks. The Katharina tunicata [Octopus vulgaris [trnP and a transposition of trnD, and differs from that of N. macromphalus by these changes plus additional transpositions of trnF and trnT and the switch in position of two large blocks of genes . This gene arrangement, plus another separately rearranged in S. officinalis, are highly derived and each shares only a few blocks of colinearity with the more conserved gene order of N. macromphalus mtDNA. All of these cephalopod mtDNAs have the same gene content except for W. scintillans and T. pacificus, the two representatives of the group Oegopsida. These two mtDNAs have a nearly identical gene arrangement, differing only in the position of trnM, that is highly rearranged from those of other mollusks, and contain duplicated copies of cox1, cox2, cox3, atp6, atp8, and trnD, such that they contain genes for a total of 18 proteins, 2 rRNAs, and 23 tRNAs. In all of these studied cephalopod mtDNAs, all genes retain the same transcriptional orientation, that is, all rearrangements are transpositions and none are inversions. Akasaki et al. [In total, there are now available complete mtDNA sequences from eight cephalopod species to compare. In addition to bleekeri , Todarodacificus ,31, WataN. macromphalus mtDNA has only one type of deviation; three genes  start with GTG and all others use the standard ATG  or TAA . In four cases  genes are probably abbreviated to a single T or to TA such that the excision of the adjacent, downstream tRNA from the polycistronic message leaves an mRNA that is polyadenylated to complete a TAA stop codon. However, in each of these cases, a complete stop codon is available if there is, alternatively, overlap of only one or two nucleotides with the downstream tRNA. Perhaps these act as a "backup" for cases where translation precedes message cleavage. The other two cases are more ambiguous. nad4L could have an abbreviated stop codon, but is inferred to overlap nad4 by seven nucleotides to the first legitimate stop codon, since overlap of this pair has been commonly observed for other mtDNAs, where they are thought to be translated as a bicistron. nad6 is inferred to overlap cob by eight nucleotides, perhaps suggesting that these are processed also as a bicistron, but could instead end on an abbreviated stop codon if there were some signal for message cleavage  that we do not recognize. Inferred in this way, all protein-encoding genes have lengths nearly identical to those of K. tunicata mtDNA  appears to overlap trnL2(yaa) by only the former's discriminator nucleotide (A). trnQ appears to overlap trnW by two nucleotides. trnK appears to overlap trnA by four nucleotides, GGCT. These are well-paired in the potential structure of tRNA(A), but these four correspond to two G-T pairs, one mismatch, and the discriminator nucleotide of tRNA(K). It appears for each case that cleavage to form a complete downstream tRNA followed by (poly)adenylation of the upstream tRNA  would yN. macromphalus mitochondrial tRNAs follow this pattern. One exception is tRNA(M), which has the anticodon CAT (to recognize both ATG and ATA), as is almost universally the case for all animal mitochondrial systems. In some cases the C is known to be post-transcriptionally modified to 5-formylcytidine to enable the necessary pairing with the ATA codon [Loligo bleekeri tRNA(S), for which the G is modified to 7-methylguanosine [Usually T is in the first anticodon position for tRNAs that recognize either four-fold degenerate codon families or to specifically recognize NNR codons; G is usually in this position only to specifically recognize NNY codons.  All but two of the TA codon . Howeveruanosine .N. macromphalus has 1,416 nucleotides that are not assigned to genes. This is not an unusually large number, but it is atypical that they are distributed among so many regions of the genome 13 run (see below), there are six repeats of a 62 nucleotide element followed by a partial repeat of 39 nucleotides. Within this are five overlapping regions that have potential for forming tRNA-like structures .In the largest non-coding region, between 3 in each of the intergenic regions of trnA-trnR and trnG-atp6 and an especially noteworthy (CA)13 in the region between trnQ and trnT. Homopolymer runs of T10, nine C9, and A20 are in the regions trnQ-trnT, trnG-atp6, and trnE-cox3, respectively. Non-coding, non-functional portions of mtDNA are generally eliminated rapidly [Tandem repeats of CA are common, with (CA) rapidly , presuma rapidly , but wheN. macromphalus mtDNA is 59.6% A+T. The strand that includes cox1, which we will arbitrarily designate as the plus strand for the purpose of discussion, is 33.7% A, 25.8% T, 11.9% G, and 28.5% C. This strand is strongly skewed  away frN. macromphalus are organized into three blocks: trnE through nad5; trnG individually; and trnQ through trnF. As can be seen in Figure The minus-strand genes of K. tunicata, contrasts with this. Although the gene arrangement is quite similar, here the pattern of bias is opposite in two different respects. First, it is the plus-strand genes that have strong skew in nucleotide composition, with the minus-strand genes being nearly neutral for this bias. Secondly, the bias for these is strongly toward G+T for the reported strand. Here again, the sharp transitions in base composition are flanked by non-coding regions at least 20 nucleotides in length, which could potentially serve as signaling elements for transcription or replication.The mitochondrial genome of the chiton, Such skews with one strand being rich in A+C and the other rich in G+T are common for mitochondrial genomes .  None of these sequences are present in K. tunicata mtDNA and none are present in the non-coding regions of any of the other studied cephalopods.An attempt was made to find potential regulatory sequence elements by comparing all pairs of non-coding regions that are greater than 13 nucleotides in length  for any N. macromphalus and each non-coding region of all of the other cephalopod mtDNAs greater than 19 nucleotides in length for any blocks of length 20 or greater matching at least 70%. Although some matches were found, none were consistent across all (or even most) species. Lastly, a search was made for all available cephalopod mtDNAs for long stretches of alternating CA or TA, suggested to play a role in regulation of replication and/or transcription. Of note is that N. macromphalus has several regions of alternating CA 4. In contrast, while N. macromphalus has no regions of alternating TA longer than (TA)3 (which occurs in eight places), each of the other cephalopods has many such regions at least of length (TA)9 (T. pacificus and W. scintillans), and some as long as (TA)22 (S. lessoniana). 14, for O. ocellatus is (TA)16, and for both S. officinalis and O. vulgaris is (TA)12.) Of course, it is possible that actual regulatory elements may be more complex and difficult to identify.A comparison was also made between each non-coding region of ating CA , the lonNautilus macromphalus is fairly typical in many respects, with a size, gene content, and A+T-richness similar to those most common for animal mtDNAs. There have been only a few gene arrangements in this lineage even since its divergence from the basal mollusk group Polyplacophora, and these rearrangements can be confidently polarized among the two lineages by comparing them to mtDNAs of less related animals.To date, complete mtDNA sequences had been determined for 23 mollusks, a very small sampling compared to those available for vertebrates or arthropods ,24, numeThere is strong skew in the distribution of nucleotides between the two strands and it appears that biases in mutational spectrum during both transcription and replication are responsible for this. Compared with most animal mtDNAs, there are a large number of non-coding regions. Although their functions, if any, are not known, the fact that several are at positions of abrupt shift in nucleotide skew and that some contain identical sequence elements suggests that they may contain regulatory signals for transcription and/or replication. This appears to be another example where polyadenylation of tRNAs creates part of the amino-acyl acceptor stem. These, and other features can be interpreted in detail for the systems of these diminutive genomes, and further sampling of complete mtDNA sequences across the tree of life promises to provide insights into general aspects of genome evolution.N. macromphalus (this 0.5% difference is presumably due to intraspecies polymorphism), whereas all others differ by from 16 to 24 positions; therefore, it appears that this sample was from N. macromphalus.Testis tissue, stored for longer than a decade at -80°C, but without any record of which species of Nautilus had been sampled, was the gift of Wesley Brown. Fortunately, GenBank contains a short fragment of mitochondrial rRNA for each of the six species of the genus, and this was used for specific identification. The 401 nucleotides in common for this sample and the GenBank records were compared to determine that only two positions differ with the record of M mannitol, 70 mM sucrose, 50 mM Tris HCl-pH 75, 3 mM CaCl2) and processed using a Tissuemizer T-25 (Tekmar) with three strokes of five seconds each. Membranes were lysed by adding 1/10 volume of 20% SDS and incubating for 20 min at RT. A 1/6 volume of saturated CsCl in water was added and this mixture incubated on ice for 15 min. Debris was pelleted at 17,000 × G for 10 minutes at 4°C. Propidium iodide was added to the collected supernatant to a final concentration of 500 μg/ml and the CsCl concentration was adjusted to a density of 1.57 g/ml. Nuclear and mitochondrial DNA were separated by density gradient centrifugation in a VTi65 rotor at 55,000 × G for 15 hours at 21°C. Although no mitochondrial band was visible in the gradient, the region from about 2–10 mm below the nuclear band was collected using a needle. This was then extracted multiple times with water-saturated butanol to remove the propidium iodide and dialyzed against TE for 24 hours with three buffer changes to remove the CsCl, leaving the sample in a 100 μl volume.Mitochondrial DNA was isolated from approximately 1 g of this tissue by first grinding in liquid nitrogen using a mortar and pestle. This powder was dissolved in 14 ml of homogenization buffer  that had been digested with EcoRV, T-tailed using Taq polymerase, and gel purified using GeneClean (QBiogene). A successful recombinant clone was selected and DNA prepared using standard techniques. The other fragments were purified by three serial passages through an Ultrafree  spin column (Millipore) and sequenced directly. The sequences of these fragments were determined using an ABI377 automated DNA sequencer with BigDye chemistry (Applied Biosystems) according to supplier's instructions.This product was used in PCR as in  to amplifound in -47. The cox1-nad1 (using conserved nad1 primer CCTGATACTAATTCAGATTCTCCTTC), nad1-cob, cob-rrnL, and rrnL – cox1 (using conserved primer 16SARL [Primers were designed to known sequences for use in long PCR  with rTtr 16SARL ), jointlK. tunicata mtDNA [Genes encoding rRNAs and proteins were identified by matching nucleotide or inferred amino acid sequences to those of ta mtDNA  through cox1,cox2,cox3, cytochrome oxidase subunit I, II, and III protein genes; cob, cytochrome b gene;atp6,atp8, ATP synthase subunit 6 and 8 genes;nad1,nad2,nad3,nad4,nad4L,nad5,nad6, NADH dehydrogenase subunit 1–6, 4L genes;trnA,trnC,trnD,trnE,trnF,trnG,trnH,trnI,trnK,trnL1(nag),trnL2(yaa),trnM,trnN,trnP,trnQ,trnR,trnS1(nct),trnS2(nga),trnT,trnV,trnW,trnY, transfer RNA genes designated by the one-letter code for the specified amino acid; in cases where there is more than one tRNA for a particular amino acid, they are differentiated by numeral and with anticodon .Gene arrangements. All available complete gene arrangements for mollusk mtDNAClick here for fileNmm. To save space the middle portions of many genes are replaced by a numeral indicating the number of omitted nucleotides. Gene orientation is specified by a dart (>). Stop codons are shown by asterisks whether complete or abbreviated, with a plus symbol indicating an alternative that overlaps the downstream gene. Down-facing arrows mark repeats found in the largest non-coding region. When not conforming to the genetic code, the presumed initiator methionine (M) is in parentheses.Gaso Click here for fileNautilus sp. and the polyplacophoran Katharina tunicataProtein lengths Comparisons of the number of amino acids in the inferred proteins between the mtDNAs of the cephalopod Click here for fileNautilus sp. mtDNAIntergenic regions Summary of the 1416 non-coding nts extracted from the intergenic regions of Click here for file


One of the goals of this study was to learn the coverage, safety and logistics of a mass vaccination campaign against typhoid fever in children and adults using locally produced typhoid Vi polysaccharide (PS) and group A meningococcal PS vaccines in southern China.The vaccination campaign targeted 118,588 persons in Hechi, Guangxi Province, aged between 5 to 60 years, in 2003. The study area was divided into 107 geographic clusters, which were randomly allocated to receive one of the single-dose parenteral vaccines. All aspects regarding vaccination logistics, feasibility and safety were documented and systematically recorded. Results of the logistics, feasibility and safety are reported.The campaign lasted 5 weeks and the overall vaccination coverage was 78%. On average, the 30 vaccine teams gave immunizations on 23 days. Vaccine rates were higher in those aged ≤ 15 years (90%) than in adolescents and young adults (70%). Planned mop-up activities increased the coverage by 17%. The overall vaccine wastage was 11%. The cold chain was maintained and documented. 66 individuals reported of adverse events out of all vaccinees, where fever (21%), malaise (19%) and local redness (19%) were the major symptoms; no life-threatening event occurred. Three needle-sharp events were reported.The mass immunization proved feasible and safe, and vaccine coverage was high. Emphasis should be placed on: injection safety measures, community involvement and incorporation of mop-up strategies into any vaccination campaign. School-based and all-age Vi mass immunizations programs are potentially important public health strategies for prevention of typhoid fever in high-risk populations in southern China. The People's Republic of China has led the use of Vi vaccine as a public health tool to contain typhoid fever in some provinces in which the disease is endemic. With Vi vaccine proven to be efficacious in clinical trials ,2, in 19Vi vaccine is currently produced by 6 vaccine institutes in China. Some provincial and district governments have encouraged its use through school-based campaigns financed with reasonable user fees . In 1996, the Guangxi Zhuang Autonomous Region (Guangxi Province) in southern China introduced Vi vaccine as a public health tool for school-aged children and for use during outbreaks. Although the reduction in typhoid fever cases from government surveillance has been reported, to date, there has been no formal assessment of the effectiveness of the Vi vaccine use as a public health tool.Guangxi Province plans to expand the Vi vaccination strategy beyond the school-aged children. An open cluster randomized controlled Vi vaccine effectiveness evaluation was designed and launched in 2002 with the goal of generating policy-relevant data to enable expanded use of Vi vaccine in public health program . Herein,The vaccination campaign was carried out in Hechi Prefecture figure , GuangxiIn recent years national child immunization program coverage has been over 90% in Hechi, which has a variety of health facilities  including hospitals, health centers, factory clinics and private clinics. The Hechi Center for Disease Control (CDC) is responsible for the collection of data on notifiable infectious diseases. In this area, in 1995–1999, typhoid fever incidence rates were 27–153 per 100,000 residents . A. A11]. ABoth vaccines used for this study are licensed and commercially available in China; however, due to the nature of research aspects in determining the effectiveness of the Vi vaccine, ethical and research clearance was obtained from the Guangxi ethical review board, the International Vaccine Institute's institutional review board and the ethical committee of the World Health Organization (WHO). Trial information was disseminated at the community level (meetings and media) and consent was recorded individually on the day of immunization.Promotional activities were initiated 2 months prior to immunization and included meetings with community leaders and campaign advertisements via local television, loudspeakers, posters and information flyers. The mass vaccination campaign was carried out in 4 stages. Stage I  vaccinated a few clusters in order to test and fine-tune the system and to provide final training for the other teams. The next stage was the large-scale vaccination at pre-defined vaccination posts in each cluster according to a pre-set schedule. In stage III, the initial mop-up, the vaccination team re-visited its corresponding vaccination post at the cluster for 1 day. The final mop-up (stage IV) was at a centralized vaccination center (Hechi CDC) after the study population was again invited to be vaccinated.Salmonella enterica subspecies enterica serovar Typhi) for both children and adults. This vaccine is administered intramuscularly. The menA vaccine vial contains five freeze-dried 0.2-ml doses (each is 30 μg of the purified group A PS of Neisseria meningitidis). Each dose must be reconstituted with 1 ml of PBS (diluent provided with the vaccine) before subcutaneous injection.Both the Vi and the menA vaccines are licensed. They are produced by Lanzhou Institute of Biological Products and administered in 1-dose regimes. A Vi vaccine vial contains two 0.5-ml doses  was given to all vaccination team members and supervisors 1 month prior to the campaign. The vaccines were delivered by methods that simulated public health program conditions. One site was selected in each cluster  to become the vaccination posts. Table Each team was expected to vaccinate about 200 persons each day and to cover 3 or 4 clusters in 20 days. Identification cards similar to those used in the local expanded program of immunization (EPI) were distributed to each household 1 month before vaccination. The cards had identifying information and a study number that corresponded to the household's date and time of vaccination. In order to avoid errors in vaccine allocation, each vaccine team delivered only one vaccine. Those registered in the project census were vaccinated and recorded. However, persons not registered in the project census were offered menA vaccine at the end of the campaign.Each vaccination team was equipped with: 1 or 2 cold boxes, injecting material, emergency kits, 1 safety disposal box and stationary. All eligible participants were listed in a vaccination record book available at the vaccine posts. Those who came for immunization and gave informed consent were assessed for eligibility by the vaccination team and vaccination status, regardless of received or not received, was recorded in the vaccination record book, which was later entered into a database. Vaccination coverage was calculated from this database.All aspects of safety for each of the study vaccines were emphasized and the vaccination teams received intensive training. Two particular aspects were monitored: safe injection practices and adverse events (AE). Proper vaccine administration (intramuscular for typhoid Vi and subcutaneous for menA), aseptic injection technique and injection safety (not recapping and disposing of used needles into safety boxes) were closely monitored by supervisors and external observers. Team members were instructed to report all accidents involving sharps and needles to their supervisor. The complete incineration of used safety boxes was monitored by the Hechi CDC staff.Safety of the vaccines was monitored systematically. All vaccinees were asked to remain in the vaccination posts for at least 15 minutes after vaccination to be monitored for any immediate serious adverse event (SAE) by the physician of the vaccination team. 535 randomly chosen vaccinees were visited for 3 consecutive days after vaccination by the project personnel for solicited adverse event surveillance. Unsolicited (passive) adverse event surveillance was carried out for one month following vaccination by having all healthcare facilities in the study area to report signs and symptoms of patients with history of receiving the vaccine, or hospitalization cases of the vaccinees to Hechi CDC. Physicians attending the patients were asked to fill in a form for all the cases with causality specified. Inadvertent vaccination of pregnant women was to be reported passively during the vaccination campaign; these cases would be followed until delivery. Deaths of study participants are being ascertained through established mortality surveillance by Hechi CDC, which gathers information from death certificates and cremation and hospital records for the 2-year follow-up period. Project personnel reviewed all incoming forms to determine the causality and when unclear, a trial clinical monitor assisted in reviewing the forms.2 room equipped with 8 refrigerators and 1 freezer. The number of vaccine vials and supplies distributed to each team was recorded daily to calculate vaccine usage and wastage. The maintenance of the cold chain was verified at all stages from the time the vaccine left the manufacturer until it reached the vaccination posts. Maximum/minimum (max/min) thermometers and battery-automated temperature recorders were used to monitor the vaccine temperature during transportation and storage. Regular thermometers in cold boxes were used at the vaccine posts. Temperatures were documented daily in temperature charts – twice for stored vaccines and 3 times for the cold boxes.For both vaccines the manufacturer recommends storage temperatures of 2–8°C. All supplies, including the vaccines, were stored at the logistic hub at Hechi CDC: a 3 × 12 mThe vaccination campaign lasted 31 workdays, during which 53 clusters received typhoid Vi vaccine and 54 the menA PS vaccine. Thirty teams gave immunizations on an average of 23 days, covering a target population of 118,588 in 107 clusters. Overall, 96,504 people came to the vaccination posts and 92,476 were immunized, yielding an overall vaccine coverage of 78%. In all, 25,605 persons were not vaccinated: 80%  did not appear for immunization. Table The vaccine coverage was 77% and 80% for Vi vaccine and menA vaccine, respectively. Figure Three needle-stick injuries were reported during the mass vaccination campaign. All cases happened to the vaccinators of the team. One case occurred during crowded hours at the vaccination post, while two cases occurred due to mishandling of the sharp disposal box. The 3 persons exposed to blood were tested for hepatitis B surface antigen (HBsAg) antibodies and immediately given 1 dose of hepatitis B vaccine according to the local guidelines. No further doses were necessary as all 3 were anti-HBsAg positive.No life-threatening immediate AE were observed. The surveillance system did identify 1 serious AE; however, this auto-limited febrile case which required hospitalization was not severe and the patient recovered in 12 hours. Of the 413 persons interviewed for solicited AE, 408 were surveyed on all 3 days, and no SAE were reported. In total 129 (38 Vi and 91 menA) AEs determined to be caused by the vaccine were reported from 66 individuals (26 Vi and 40 menA), consisting mainly in fever , malaise (19% Vi and 10% menA), and/or minor rash (19% Vi and 5% menA) around the site of injection. In all, 3,430 hospitalizations were reported 1 month after the immunizations. Of these persons, 300 had been immunized during the campaign. The trial clinical monitor determined that none of the hospitalizations (the majority were scheduled surgeries) were related to vaccine injections.The Lanzhou Institute initially shipped 60,000 doses of Vi vaccine and 80,000 doses of menA vaccine to the study site by refrigerated (2–4°C) truck monitored by max/min thermometer. Once the temperature reached 14°C for approximately 3 hours. The vaccines were stored in cold rooms at the Guangxi CDC for 6 days where temperature ranged from 2°C to 8°C. On March 29, 2003, the vaccines were sent to the Hechi CDC in a refrigerated truck. This 420-km journey took 6 hours. The cold chain (monitored by battery-powered temperature recorder) was maintained between 4°C and 9°C. At the Hechi CDC, all 140,000 vaccine doses had temperature readings of 2°C to 11°C. The mean temperature recorded in the field was 4.2°C (range: 1.8°C – 12°C). In all, 26 vials of Vi vaccine were discarded due to freezing.Wastage based on breakage, missing at inventory and unused opened vials was higher as expected for the 5-dose vial of the menA vaccine vial than for the Vi vaccine . In conformity with the product information sheet, vials of menA vaccine were discarded within 1 hour of opening if not used. At the end of the campaign the unused doses of both vaccines were shipped to the Hechi CDC headquarters so they could be used elsewhere.The number of personnel required during the campaign is shown in table The results verify that a mass vaccination campaign with the 1-dose parenteral Vi vaccine or the menA vaccine, which targeted both school-aged children and adults in Hechi, south China, was logistically feasible and safe. An important coverage rate was attained with no major disruption to the normal EPI or other health activities.In general, there are three main concerns when launching population-based large-scale vaccination campaigns: (1) resources, (2) safety and (3) cost. Especially in developing countries, there is the need to identify resources outside the well-established local vaccine delivery system . The Hechi campaign illustrates that an existing healthcare system can deliver vaccines to large populations in a fairly short period of time. Community members can play a key role in the success of promotional activities and delivery of vaccines when an entire city is targeted.The Hechi campaign monitored and recorded safety systematically and in depth. No life-threatening serious AE were associated with either vaccine. No reported AE (solicited or not) were serious and all patients recovered quickly. Use of safety boxes was introduced for the first time in Hechi during this mass vaccination campaign. Despite instruction on safe handling of sharps, 3 needle-stick injuries were recorded. For future mass vaccination campaigns, including routine EPI activities, safe injection practices must be introduced and emphasized to prevent unnecessary accidents and spread of blood-borne diseases.A mop-up strategy increased the coverage by 9.6% on average in the lower-coverage clusters and by 0.7% increase in the higher-coverage clusters. Because mop-up strategies imply the need for additional time and costs, this strategy should be planned in advance. Adult coverage was slightly lower than that of children, but a 70% coverage indicates the ability to reach adults during a vaccination campaign. The comprehensive vaccine safety surveillance system, implemented within the existing Hechi health facilities, was useful. Post-licensure monitoring can address many issues regarding safety, such as: uncommon AE, incorrect storage or administration procedures and dissemination of information to the public to maintain confidence in the scheme .The cost-effectiveness of the use of Vi vaccine will be analyzed at the end of the study. However, because the vaccine is locally produced, it seems reasonable to anticipate that unlike EPI vaccines, cost savings can be achieved by reduced transportation costs and in-country production of the vaccine. In an urban area of Vietnam, a locally produced oral cholera vaccine distributed through mass immunization was found to be affordable if introduced in a public health program . In counMass immunization is considered the most logical control strategy, outside the EPI program, in settings that typically manifest high incidences of disease or in populations exposed to predictable outbreaks . BesidesThe author(s) declare that they have no competing interests.YJ, CJA, MA, YH, TP, XZY, AD, DB, JDC have been in part of the conception and design of study, YJ, CJA, SG, ZengJ, LC, LD, RLO, AP, MCD, ZhangJ, ZB, LH, WM, TD, TZ, GJ, JKP, MA, BI, LG, YH, TP, XZY, AD, CMG, DB, JDC compiled, analyzed, and/or interpreted the data, and YJ, CJA, SG, ZengJ, LC, LD, RLO, AP, MCD, ZhangJ, ZB, LH, WM, TD, TZ, GJ, JKP, MA, BI, LG, YH, TP, XZY, AD, CMG, DB, JDC played roles in drafting and/or revising the manuscript.The pre-publication history for this paper can be accessed here: