Nested Polymerase Chain Reaction-restriction Fragment Research Articles

Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle

A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1β gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1β and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1β amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1β sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated.

Genotyping of Cryptosporidium species in children suffering from diarrhea in Sharkyia Governorate, Egypt.

The protozoan parasite Cryptosporidium is one of the principal reasons for childhood diarrhea around the world. This work aimed to differentiate Cryptosporidium species among children suffering from diarrhea in Sharkyia Governorate, Egypt. A total of 97 fecal specimens were taken from children suffering from diarrhea, attending Pediatric Clinics of Zagazig University and Al-Ahrar Hospitals. Full history was taken. Stool samples were examined microscopically using modified Ziehl-Neelsen stain for detection of Cryptosporidium oocysts. To identify Cryptosporidium genotypes, positive samples were then subjected to nested Polymerase chain reaction-restriction fragment length polymorphism targeting Cryptosporidium oocyst wall protein gene. The overall detection rate was 27.8% (27/97) using modified Ziehl-Neelsen stain staining method. Using nested polymerase chain reaction, the gene was amplified in 85.2% (23/27). Restriction fragment length polymorphism analysis revealed that 65.2% (15/23) were Cryptosporidium hominis, 30.4% (7/23) were Cryptosporidium parvum, and one sample was not typed (4.4%). The significant risk factors associated with Cryptosporidium infection in children were animal contact and residence in rural areas. Cryptosporidium is a common enteric parasite affecting children in Sharkyia Governorate, Egypt, with the predominance of C. hominis genotype in children.

Molecular prevalence of Cryptosporidium isolates among Egyptian children with cancer.

Immunocompromised individuals especially children with cancer are at risk for acquiring cryptosporidiosis, which can result in severe morbidity and mortality. This work was conducted to evaluate the prevalence of Cryptosporidium parasite and its genotypes in children with cancer. Stool specimens were collected from 145 children in the Oncology unit of Pediatric Department, Zagazig University Hospital, Sharqiyah province, Egypt. Cryptosporidium infection was evaluated using modified Ziehl-Neelsen (MZN) staining and nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The prevalence of Cryptosporidium infection in oncological children was 29.7% using microscopy and 25.5% using nested PCR. Genotypic characterization showed that 23 (62.2%) had C. hominis, 11 (29.7%) C. parvum, and 3 specimens (8.1%) were mixed infection of both genotypes. Cryptosporidiosis was significantly associated with diarrhea. However, no statistically significant difference was detected between the age, gender, residency, animal contact and malignancy type concerning to Cryptosporidium infection. This study concluded that Cryptosporidium is a prevailing opportunistic parasite among children with cancer. It should be considered in oncological patients especially those suffering from diarrhea which requires proper management to reduce its complications.

An Integrated Study of Toxocara Infection in Honduran Children: Human Seroepidemiology and Environmental Contamination in a Coastal Community.

(1) Background: Infections caused by Toxocara canis and T. cati are considered zoonoses of global importance. Reports from North and South America indicate that human infections are widespread in both continents, but epidemiological information from Central America is still lacking. (2) Methodology: In the present cross-sectional multi-year study, we aimed to undertake the first seroepidemiological and environmental study on toxocariasis in Honduras. This included the determination of seroprevalence of anti-Toxocara spp. antibodies in children using a Toxocara spp. purified excretory-secretory antigens enzyme-linked immunosorbent assay (TES-ELISA) and a confirmatory Western blot. As well, through statistical analysis including logistic regression we aimed at identifying relevant biological and epidemiological factors associated with seropositivity. The study also entailed detection of parasites’ eggs in the soil samples both through Sheather’s concentration method and a nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. (3) Results: The study was undertaken in a coastal community of Honduras in 2 different years, 2015 and 2017. A total of 88 healthy schoolchildren completed the study, with participation of 79% (73/92) and 65% (46/71) of the student body in 2015 and 2017, respectively. Thirty-one children participated in both years (i.e., dual participants). Through both serological tests, seropositivity was confirmed in 88.6% (78/88) of children. Due to the high number of seropositives, logistic regression analysis was not possible for most socio-economic and epidemiological variables. Eosinophilia, on the other hand, was associated with seropositivity, independently of other intestinal helminthic infections. Continued seropositivity was observed in most of the dual participants, while seroconversion was determined in 8 of these children. Microscopic examination of soil samples did not yield any positive results. Through nested PCR-RFLP, 3 of the 50 samples (6%) were positive for Toxocara spp.; two were identified as T. canis and one as T. cati. (4) Conclusions: This work documents for the first time, high levels of human exposure to Toxocara spp. in Honduras. These findings, along with the country’s favorable epidemiological conditions for this zoonosis, emphasize the need for more research to determine whether this infection is underreported in the country.

Molecular genetic characterization of human Cryptosporidium isolates and their respective demographic, environmental and clinical manifestations in Egyptian diarrheic patients

Background: Cryptosporidium is a zoonotic protozoan parasite of public health and veterinary importance, causing gastroenteritis in a variety of vertebrate hosts. Several Cryptosporidium spp. with different genotypes and subtypes are implicated in human cryptosporidiosis. Objective: Detection and correlation of native Cryptosporidium molecular genetic variability among Egyptian diarrheic human isolates with their respective demographics, environmental and clinical manifestations. Patients and Methods: A total of 350 human stool samples were collected from diarrheic patients. The collected samples were examined microscopically and by RIDA®QUICK Cryptosporidium/Giardia copro-immunochromatography test (ICT) cassettes and strips. All positive Cryptosporidium samples detected by modified Ziehl-Neelsen stain (MZN) and/or ICT were genotyped using nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) targeting cowp gene encoding Cryptosporidium oocyst wall protein ( Cowp). Results: Cryptosporidium was detected in 5.7% (20/350) of the examined samples using MZN, 9.4% (33/350) by ICT kit, and 3.7% (13/350) by both methods. A total of 40 samples were positive by both microscopy and/or ICT. Only 15/40 (37.5%) of samples positive by microscopy and/or ICT were successfully amplified using cowp gene. All amplified samples (15) were confirmed as C. hominis by nPCR-RFLP analysis of the cowp gene. Significant associations were found between cryptosporidiosis and gender, drinking groundwater, immunodeficiency of patients, complaining of fever, watery and mucoid stool, consumption of readymade salad, and unwashed fresh vegetables. Conclusion: In the current examined samples, C. hominis proved to be the dominant genotype, indicating that transmission of cryptosporidiosis among a sample of diarrheic Egyptian patients is most probably anthroponotic rather than zoonotic. MZN and PCR gave a diagnostic accuracy of 100%, while ICT diagnostic accuracy proved to be 50%.

Toxoplasma gondii genotypes circulating in domestic pigs in Serbia

Consumption of undercooked or raw pork is considered a significant risk factor for human infection with Toxoplasma gondii. In this study, we investigated the genetic structure of 18 T. gondii strains obtained from slaughter pigs from Northern Serbia (mainly Vojvodina). The examined samples originated from eight pigs from large commercial farms, six backyard pigs and four free-range Mangalica pigs, all found to be positive for either viable T. gondii or T. gondii DNA. Genotyping was attempted from both pig tissues and mouse brains from the bio-assays using a multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP) method with seven markers (GRA6, alt. SAG2, PK-1, BTUB, C22-8, CS3 and Apico). Identification was achieved for nine T. gondii isolates. Seven isolates were classified as type II and two as type III. These results are consistent with previous studies on animal isolates from Serbia as well as with previous reports that type III is more frequently found in samples from Southern Europe than in those from other parts of the continent.

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.

Insight into epidemiological importance of phytoplasma vectors in vineyards in South Moravia, Czech Republic

Bois noir (BN), caused by ‘Candidatus Phytoplasma solani’, is a serious disease of grapevines in Europe. During the 2010–2012 survey in Perná vineyard (South Moravia, Czech Republic) a total of 4854 insect individuals were collected and among these, 95 insect species belonging to Auchenorrhyncha (77 species), Heteroptera (12), and Psylloidea (62) were indentified. The nested polymerase chain reaction-restriction fragment length polymorphism analyses confirmed Hyalesthes obsoletus as the main BN vector with 43.8% of phytoplasma positive individuals on average. A significant role of Anaceratagallia ribauti (22.6% of phytoplasma positive specimens) should be taken into account based on its occurrence and incidence of infected individuals. Eleven insect species were identified as new carriers of ‘Ca. P. solani’ or suggested as potential BN vectors in this work.

Preliminary report: one of the PD-1 gene variants may be a valuable marker for colorectal cancer

IntroductionIntroduction: Programmed death-1 (PD-1), an important immunosuppressive molecule, plays a key role in tumor-cell-mediated immune escape. The present study aimed to investigate the role of PD-1.5 (C/T) gene polymorphisms on the susceptibility and progression of colorectal cancer (CRC).Material and methodsIn this study, the PD-1.5 C/T polymorphism was investigated in 99 CRC patients and 150 healthy individuals as controls by nested polymerase chain reaction-restriction fragment length polymorphism methodResultsThe distributions of PD-1.5 (C/T) genotypes and alleles were in agreement with Hardy–Weinberg equilibrium in controls (p>0.05) but not in CRC patients (p=0.02). We found a statistical significance difference between CRC patients and controls for the genotypic distribution of PD-1.5(C/T) genotypes (p=0.003) and also for alleles (p=0.004). The patients who have T allele have increased according to the controls (p=0.001). The patients who have C allele with distance metastasis have increased heterozygous CT genotype of PD-1.5 (C/T) polymorphism than those with no metastasis (p<0.001). We also detected the increased CC genotype in patients who have angiolymphatic invasion (p=0.043). The patients who have mucineous component have increased frequency of T allele than those with the absence of mucineous component (p=0.023).ConclusionsOur results have shown significant associations between PD-1.5 genotypes and CRC susceptibility and progression of the disease.

Molecular detection and genetic characterization of Toxoplasma gondii in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong province, eastern China

Toxoplasma gondii is a successful opportunistic parasite, affecting a wide range of vertebrate animals and humans. Genetic diversity of T. gondii in raccoon dogs (Nyctereutes procyonoides) is of great importance to understand the transmission of T. gondii in the environment. However, no information is available about the distribution of genetic diversity of T. gondii infection in raccoon dogs. This study was conducted to estimate the prevalence and genetic characterization of T. gondii from raccoon dogs in Shandong province, eastern China. A total of 314 brain tissue samples of raccoon dogs were collected and genomic DNA was extracted and assayed for T. gondii infection using semi-nested PCR targeting B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2(5′+3′ SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) by multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn- PCR-RFLP) technology. Thirty-five (11.15%) of 314 DNA samples were detected positive. Only six samples were completely typed at all genetic loci, and these samples represented ToxoDB genotype#9. Two sample were typed at 9 genetic loci and one was typed at 8 genetic loci, all of them represented Type I. To our knowledge, this is the first report of genetic characterization of T. gondii in raccoon dogs in China. These results revealed the existence of genetic diversity of T. gondii in raccoon dogs in China. These data provided base-line information for controlling T. gondii infection in raccoon dogs.

Isolation and characterization of Toxoplasma gondii from small ruminants (sheep and goats) in Chennai City, South India.

The present study aimed for the isolation and genotyping of Toxoplasma gondii from small ruminants (sheep and goats). 14 out of 193 tissue samples (either brain and heart) tested positive by MDAT for anti-T. gondii antibodies, were selected and bioassayed, which resulted 4 samples positive for T. gondii after 40days of post inoculation. Four samples consisting of 3 numbers of sheep and 1 number of goat tissues out of 14 samples detected by B1 PCR, were genotyped at SAG3 locus by nested polymerase chain reaction-restriction fragment length polymorphism technique (nPCR-RFLP). The results of the present study revealed that the four isolates designated as TgShIn19, TgShIn76, TgShIn77 and TgGtIn27 were circulating in small ruminants, were belonged to genotypes of type II (TgShIn19) and type III (TgShIn76, TgShIn77 and TgGtIn27) which are in concordance with the previously reported genotypes from other animal species and further this presumptive results indicating that the genotype II and III could be the predominant in different animal species including birds and humans in India.

Genetic and Environmental Biomarkers Associated with Triglyceride Levels in Two Groups of Slovak Women.

This study analyzed the association between the MLXIPL gene polymorphism (rs3812316) and triglyceride (TG) levels and selected environmental biomarkers in Slovak women at risk for cardiovascular disease compared to a reference sample. The studied sample consisted of 200 women at cardiovascular risk (mean age 52.96 ± 6.01 years) and 244 healthy women (mean age 47.52 ± 5.34 years). Participants gave details of their health and lifestyle during their medical examination, and peripheral blood samples were used for biochemical analyses and DNA genotyping. A nested polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the rs 3812316 SNP. We determined that there were significantly different genotype distributions in two TG categories: (1) subjects with normal TG values had a significantly higher G allele frequency than those with elevated TG levels (χ2 = 6.1556, df = 2, p = 0.046); and (2) the rare G allele frequency was 0.11 in the cardiovascular risk group and 0.15 in the reference group. Binary regression analysis showed that women with at least one G allele had a significantly lower relative risk of hypertriglyceridemia than women with the CC genotype (OR = 0.399, p = 0.022, 95% CI = 0.182-0.876). This cross-sectional study suggests that MLXIPL rs3812316 genotypes may be associated with TG levels. However, further analysis is advisable because of study limitations.

Plasmodium vivax msp-3α polymorphisms: analysis in the Indian subcontinent.

BackgroundPlasmodium vivax is the most widely distributed human malaria parasite and accounts for approximately the same number of malaria cases as Plasmodium falciparum in India. Compared with P. falciparum, P. vivax is difficult to eradicate because of its tendency to cause relapses, which impacts treatment and control strategies. The genetic diversity of these parasites, particularly of the merozoite surface protein-3 alpha (msp-3α) gene, can be used to help develop a potential vaccine. The present study aimed to investigate the genetic diversity of P. vivax using the highly polymorphic antigen gene msp-3α and to assess the suitability of using this gene for population genetic studies of P. vivax isolates and was carried out in 2004–06. No recent study has been reported for MSP 3α in the recent decade in India. Limited reports are available on the genetic diversity of the P. vivax population in India; hence, this report aimed to improve the understanding of the molecular epidemiology of the parasite by studying the P. vivax msp-3α (Pvmsp-3α) marker from P. vivax field isolates from India.MethodsField isolates were collected from different sites distributed across eight states in India. A total of 182 blood samples were analysed by a nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique using the HhaI and AluI restriction enzymes to determine genetic msp-3α variation among clinical P. vivax isolates.ResultsBased on the length variants of the PCR products of Pvmsp-3α gene, three allele sizes, Type A (1.8 kb), Type B (1.5 kb) and Type C (1.2 kb) were detected among the 182 samples. Type A PCR amplicon was more predominant (75.4 %) in the samples compared with the Type B (14.3 %) and Type C (10.0 %) polymorphisms. Among all of the samples analysed, 8.2 % were mixed infections detected by PCR alone. Restriction fragment length polymorphism (RFLP) analysis involving the restriction enzymes AluI and HhaI generated fragment sizes that were highly polymorphic and revealed substantial diversity at the nucleotide level.ConclusionsThe present study is the first extensive study in India using the Pvmsp-3α marker. The results indicated that Pvmps-3α, a polymorphic genetic marker of P. vivax, exhibited considerable variability in infection prevalence in field isolates from India. Additionally, the mean multiplicity of infection observed at all of the study sites indicated that P. vivax is highly diverse in nature in India, and Pvmsp-3α is likely an effective and promising epidemiological marker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1524-y) contains supplementary material, which is available to authorized users.

Contribution of IL6 -174 G>C and IL1B +3954 C>T polymorphisms to congenital infection with Toxoplasma gondii.

The purpose of this investigation was the determination of the distribution of genotypes and alleles, residing within interleukin 6 (IL6) and interleukin 1 (IL1) polymorphisms, among fetuses and neonates, congenitally infected with Toxoplasma gondii, and among uninfected control cases. The study included 22 fetuses and newborns infected with T. gondii and 49 control cases. Screening for IgG and IgM antibodies against the parasite and IgG avidity was performed by enzyme-linked fluorescent assay (ELFA) tests. Quantitation of T. gondii DNA in amniotic fluids was assayed by the real-time Q PCR technique for the parasitic B1 gene. Genotypes at IL6 and IL1 single nucleotide polymorphisms (SNPs) were determined by a self-designed, nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Representative genotypes at the studied loci were confirmed by sequencing. All the genotypes were estimated for Hardy–Weinberg equilibrium and IL1 genotypes were tested for linkage disequilibrium. Genotypes and haplotypes at the studied SNPs were investigated for their possible association with the occurrence of congenital T. gondii infection, using a logistic regression model. GC heterozygotes at the IL6 −174 G>C SNP were significantly associated with toxoplasmosis and increased the risk of T. gondii infection [odds ratio (OR) 4.24, 95 % confidence interval (CI) 1.24–14.50 in the codominant model, p ≤ 0.050]. In case of IL1 SNPs, similar prevalence rates were observed between T. gondii-infected and -uninfected offspring. Regarding allelic variability, the C alleles at both IL6 and IL1B SNPs were significantly more frequent in the infected than in the uninfected cases (p ≤ 0.050). It is concluded that IL6 −174 G>C and IL1B +3954 C>T SNPs might be involved in the development of congenital T. gondii infection.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation.

To develop a Fok-I nested polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) method for the detection of hepatitis B virus X region (HBx) V5M mutation. Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma (HCC) and 77 carrier patients]. To identify V5M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I (GGA TGN9↓) was done. For size comparison, the enzyme-treated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. The assay enabled the identification of 69 patients (sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5M prevalence in HCC patients was significantly higher than in carrier patients (47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBxAg V5M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections. The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBxAg V5M mutation in chronic patients with genotype C2 infection.

Programmed death-1 gene polymorphism (PD-1.5 C/T) is associated with colon cancer

Programmed death-1 (PD-1), expressed by activated T cells, is a negative regulator of T lymphocytes. The associations of the immune response-related genes with cancer have been demonstrated. In this study, the PD-1.5 C/T (+7785) polymorphism was investigated in 200 colorectal cancer patients and 200 healthy individuals as controls by nested polymerase chain reaction-restriction fragment length polymorphism method. The genotype and allele frequencies at PD-1.5 position were not significantly different between control individuals and the overall colorectal cancer patients. However, subdivision of the patients by the location (175 colon cancer and 25 rectal cancer) revealed a significant difference between colon cancer patients and healthy individuals (p=0.026), and between colon and rectal cancer patients (p=0.017). The frequency of the CT genotype was significantly higher in colon cancer patients than in control individuals (58.3% vs. 44.8%, Bonferroni corrected p-value=0.024; OR=1.74; 95% CI=1.15–2.62), and in rectal cancer patients (58.3% vs. 28.0%, Bonferroni corrected p-value=0.012; OR=3.59; 95% CI=1.42–9.04). Characteristics of the patients including age, sex, tumor grade and stage were not associated with the PD-1.5 polymorphism. Our results show a significant association between PD-1.5 polymorphism and colon cancer. Larger numbers of patients are required to investigate comprehensively the association of rectal cancer with PD-1.5 polymorphism.

Cross-Reactivity of Some &lt;i&gt;Cryptosporidium&lt;/i&gt; Species with &lt;i&gt;Cryptosporidium parvum&lt;/i&gt; Coproantigen in a Commercial ELISA Kit

To obtain information about the prevalence of Cryptosporidium parvum in cattle in Oyo State, Nigeria, a commercially available enzyme-linked immunosorbent assay (ELISA) kit made from Cryptosporidium-specificantibodies raised against antigens of Cryptosporidium parvum was used to screen 406 randomly collected fecal samples from cattle in four White Fulani herds. The overall prevalence was 32.3% (131/406) with 44 % (37/84), 36.5% (35/96) and 26.1% (59/226) prevalence in ages 12 months respectively. Further analysis of some of the ELISA-positive samples using nested polymerase chain reaction-restriction fragment length polymorphism (PCR– RFLP) and DNA sequencing of the small subunit (18S) rRNA gene identified the isolates asCryptosporidium bovis and C. ryanae. The use of molecular tools showed that there is crossreactivity between C. bovis and C. ryanae with theCryptosporidium-specific antibodies raised against antigens of Cryptosporidium parvum, thereby eliminating the false alarm about the possible risk of zoonotic transmission in the study area. This work showed that unlike some other livestock diseases where commercially available ELISA kits are often relied on as diagnostic tools, ELISA kits obtainedfrom Cryptosporidium-specific antibodies are not reliable for assessing risk of zoonosis in epidemiological studies.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Rapid quantification and analysis of genetic diversity among Gordonia populations in foaming activated sludge plants

Activated sludge plants, sporadically suffers malfunction due to the proliferation of filamentous bacteria mainly Gordonia and Microthrix species. Nested Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (nested PCR-RFLP) in combination with quantitative real-time PCR (q PCR) was applied to study the distribution of Gordonia in foaming samples. Samples of mixed liquor were collected from three full-scale activated sludge plants that were experiencing filamentous biological foaming. Partial sequencing of 16S rRNA genes revealed the dominance of Gordonia amarae (60-80%), Gordonia terrae (10%), Gordonia polyisoprenivorans (30-40%) and an unidentified Gordonia species (20-50%). Restriction enzyme analysis of the amplicons exhibited 87.14 to 99.6% similarity at interspecies level. The q PCR results showed that there was an average of 15.6% Gordonia 16S rRNA copies with respect to the total bacterial 16S rRNA gene in foaming sludge samples with the highest being 23.51% and lowest being 10.28%. The presence of Gordonia spp. in the foaming samples was observed throughout the year but was lower during winter and its presence was significantly higher in foaming samples, compared to Microthrix parvicella (r = 0.007, P < 5%). This approach could help to quantify and confirm the existence of genetically diverse indigenous Gordonia spp. in foaming samples and can be used as an indicator of forthcoming foaming incidents.

Program death 1 (PD1) haplotyping in patients with breast carcinoma

Located on chromosome 2q37.3, the programmed death 1 (PD1) gene encodes for PD-1 (also known as CD279), a negative co-stimulator in the immune system. PD-1 renders potent inhibitory effects on T and B lymphocytes as well as monocyte responses. Expression of PD-1 ligands by tumor cells has been reported to contribute in immune system evasion. We aimed, in current study, to investigate the association of two single nucleotide polymorphisms in PD1 gene, +7146 G to A (PD-1.3) and +7785 C to T (PD-1.5 or +872), with susceptibility and/or progression of breast carcinoma. Four hundred forty-three women with breast cancer and 328 age-sex match healthy donors were recruited in present study. Genotyping was performed using Nested polymerase chain reaction-restriction fragment length polymorphisms. Arlequin software package was used to check for the Hardy-Weinberg equilibration and to determine the haplotypes. Results revealed no significant differences in the frequencies of genotypes and alleles at PD-1.3 (P=0.252 and 0.279 for genotypes and alleles, respectively) and PD-1.5 positions (P=0.522 and 0.278 for genotypes and alleles, respectively). Four haplotypes were observed among populations with no differences in the frequency between patients and controls. Our results also revealed no association between PD1 genotypes and tumor stage, tumor size, tumor grade, lymph node involvement, vascular invasion, distant metastasis, and Nottingham prognostic index. Present data do not confirm association of PD-1.3 (+7146) G/A and PD-1.5 (+7785 or +872) C/T genetic markers with susceptibility of Iranians to breast cancer.