Mismatch Amplification Mutation Assay Research Articles

The effectiveness of an SNP marker in the cholecystokinin type A receptor gene for improving growth traits in Amakusa Daioh cross chickens

Amakusa Daioh cross chickens from Kumamoto Prefecture, Kyushu Island, Japan, are F 1 hybrid chickens resulting from a cross between the Amakusa Daioh and Kyushu Rhode breeds. A significant association has been reported between a SNP (AB604331, g.420 C>A) in the cholecystokinin type A receptor gene and growth traits in some Japanese slow-growing chickens. This genetic improvement effect was examined in Amakusa Daioh cross chickens by comparing superior A allele-fixed chickens with conventional ones. Fattening experiments using 436 conventional and 297 improved (A allele-fixed) Amakusa Daioh cross birds were performed over two seasons. In each season, experimental birds of both sexes were hatched on the same day, raised in the same chicken house, and fed the same diet ad libitum for 15 wk for cockerels and 19 wk for pullets, after which they were slaughtered. Body weight (BW), average daily gain ( ADG ), and carcass weights were measured. SNP genotypes were determined using mismatch amplification mutation assay. Association between the SNP and growth traits was analyzed using GLM, taking into account gender, season, genetic additive and dominance, and test group effects. The improved pullets were significantly heavier than the conventional pullets at slaughter age. Boneless carcasses of the improved pullets were significantly heavier than those of the conventional pullets as well. In the conventional cockerels, significantly positive additive effects of the A allele on BW were detected during the entire experimental period, except at 10 and 11 wk. Significantly positive additive effects of the A allele on thigh weight were detected. We concluded that g.420 C>A is a good selective marker for increased slaughter live weight and meat weight in Amakusa Daioh cross chickens.

Tracking the Emergence of Azithromycin Resistance in Multiple Genotypes of Typhoidal Salmonella.

ABSTRACTThe rising prevalence of antimicrobial resistance in Salmonella enterica serovars Typhi and Paratyphi A, causative agents of typhoid and paratyphoid, have led to fears of untreatable infections. Of specific concern is the emerging resistance against azithromycin, the only remaining oral drug to treat extensively drug resistant (XDR) typhoid. Since the first report of azithromycin resistance from Bangladesh in 2019, cases have been reported from Nepal, India, and Pakistan. The genetic basis of this resistance is a single point mutation in the efflux pump AcrB (R717Q/L). Here, we report 38 additional cases of azithromycin-resistant (AzmR) Salmonella Typhi and Paratyphi A isolated in Bangladesh between 2016 and 2018. Using genomic analysis of 56 AzmR isolates from South Asia with AcrB-R717Q/L, we confirm that this mutation has spontaneously emerged in different Salmonella Typhi and Paratyphi A genotypes. The largest cluster of AzmR Typhi belonged to genotype 4.3.1.1; Bayesian analysis predicts the mutation to have emerged sometime in 2010. A travel-related Typhi isolate with AcrB-R717Q belonging to 4.3.1.1 was isolated in the United Kingdom, increasing fears of global spread. For real-time detection of AcrB-R717Q/L, we developed an extraction-free, rapid, and low-cost mismatch amplification mutation assay (MAMA). Validation of MAMA using 113 AzmR and non-AzmR isolates yielded >98% specificity and sensitivity versus phenotypic and whole-genome sequencing assays currently used for azithromycin resistance detection. With increasing azithromycin use, AcrB-R717Q/L is likely to be acquired by XDR strains. The proposed tool for active detection and surveillance of this mutation may detect pan-oral drug resistance early, giving us a window to intervene.

Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates.

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102−104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns.

Identification of a Mycobacterium tuberculosis-specific gene marker for diagnosis of tuberculosis using semi-nested melt-MAMA qPCR (lprM-MAMA)

Tuberculosis (TB) is the deadliest of infectious diseases. TB diagnosis, based on sputum microscopy, culture, and nucleic acid amplification tests (NAATs) to identify its main causative agent, Mycobacterium tuberculosis (MTB), remains challenging. The current available NAATs, endorsed by World Health Organization (WHO), can differentiate MTB from some MTB complex (MTBC) members. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in lprM (Rv1970) gene that differentiate MTB from other MTBC members. A forward mismatch amplification mutation assay (MAMA) primer was designed for the targeted mutation and was used in a semi-nested melt-MAMA qPCR (lprM-MAMA). Using the optimized protocol, lprM-MAMA was positive with all MTB reference and clinical strains, and negative with other MTBC members, non-tuberculous mycobacteria (NTM) and other non-mycobacterial (NM) reference strains. The limit of detection (LOD) of lprM-MAMA was 76.29 fg. Xpert® MTB/RIF (Xpert)-positive sputum samples were also positive by lprM-MAMA, except for samples classified as having “very low” bacterial load by Xpert. Xpert-negative sputum samples were also negative by lprM-MAMA. In conclusion, lprM-MAMA demonstrated to be a useful tool for specific MTB diagnosis. Further evaluation with higher number of reference strains, including NTM and NM; and sputum samples are required to determine its potential for clinical application.

Development of molecular biological tools for the rapid determination of antibiotic susceptibility of Mycoplasma hyopneumoniae isolates

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, a contagious respiratory disease, causing significant economic losses worldwide. Antibiotic treatment is commonly utilised in the pig industry to control M. hyopneumoniae infection. Since the conventional antibiotic susceptibility test is time-consuming, taking up to weeks’ period, antibiotics are usually empirically chosen.Certain single nucleotide polymorphisms in the parC (C239A/T, G250A) and gyrA (G242C, C247 T, A260 G) genes show correlation with decreased fluoroquinolone susceptibility by the change of the target site. Furthermore, the nucleotide alteration A2059 G in the 23S rRNA sequence correlates with significantly decreased macrolide and lincosamide susceptibility of M. hyopneumoniae. Mismatch amplification mutation assays (MAMA) and high resolution melt (HRM) analysis, capable to detect the mentioned resistance markers, were developed in the present study, in order to provide susceptibility data in a considerably shorter time than the conventional methods. The results of the MAMA and HRM assays were congruent with the results of the conventional antibiotic susceptibility method of the tested M. hyopneumoniae field isolates. The sensitivity of the MAMAs was 103-104 copy numbers, while that of the HRM assay was 105-106 copy numbers.To the best of our knowledge this was the first time that MAMA and HRM assays were developed for the rapid detection of decreased fluoroquinolone, macrolide or lincosamide susceptibility in M. hyopneumoniae strains.

Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.

Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates

ABSTRACT Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Verification of the Effectiveness of an SNP Marker in the Cholecystokinin Type A Receptor Gene for Improving Growth Traits in Okumino-kojidori Chickens.

A significant association was reported between a single nucleotide polymorphism (SNP; AB604331, g.420 C>A) in the cholecystokinin type A receptor gene and growth traits in some Japanese slow-growing chickens. Demonstration tests of the genetic improvement effect by comparing the superior allele-A fixed chickens with conventional ones were carried out considering the effect of different seasons on growth traits in other slow-growing chickens. Meat-type Okumino-kojidori chickens from Gifu Prefecture are a three-way cross of Gifu-jidori improved, White Plymouth Rock, and Rhode Island Red breeds. We used a total of 468 meat-type Okumino-kojidori: 264 individuals from a private hatchery as conventional chickens and 204 A-allele fixed individuals from the Gifu Prefectural Livestock Research Institute as improved chickens. We performed fattening experiments over two seasons: summer and winter. In each season, experimental birds of both sexes were hatched on the same day, raised in the same chicken house, and fed the same diet ad libitum for 12 weeks. Body weight was recorded at 3, 6, 9, and 12 weeks of age. SNP genotypes were determined using the mismatch amplification mutation assay. Association between the SNP and growth traits was analyzed using generalized linear models built on sex-based, seasonal, additive, and dominance genetic effects. The observed AA, AC, and CC genotype frequencies in the conventional chickens were 0.158, 0.479, and 0.363, respectively; body weight at 12 weeks and average daily gain from 3 to 12 weeks was superior for the A allele compared to the C allele. The improved chickens were heavier than the conventional ones at 12 weeks. Body weight at 12 weeks in allele-A fixed chickens increased by 3.2% compared to the conventional chickens. We concluded that g.420 C>A is a good selective marker that increases slaughter weight in the meat-type Okumino-kojidori chickens.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

The A Allele of the Cholecystokinin Type A Receptor Gene g.420 C > A Polymorphism Improves Growth Traits in Amakusa Daioh Cross Chicken.

Amakusa Daioh cross chickens are F1 hybrids of restored Amakusa Daioh sires and Kyushu Rhode dams. In the present study, the association between a single nucleotide polymorphism (SNP; AB604331, g.420 C>A) in the cholecystokinin type A receptor gene and growth traits in Amakusa Daioh cross chicken were investigated. We used 72 male and 72 female birds that had hatched on the same day, were raised in the same chicken house, and were fed the same diet ad libitum from day 0 to 17 weeks (wks) of age. Body weight was recorded at weekly intervals and average daily gain of each week interval was calculated from body weight data. Birds were sacrificed at 17 wks and carcass traits were recorded. SNP genotyping was carried out using the mismatch amplification mutation assay. Associations between the SNP and growth traits were analyzed by a generalized linear model. Body weight from 6 to 17 wks was higher in birds with the A allele than in birds with the C allele, although significant differences in average daily gain traits between birds with A and C alleles were not detected during most of the duration of the experiment. Carcass data showed that birds with the A allele had heavier wings and a smaller proportion of the gizzard than those with the C allele. The g.420 C>A SNP will be useful as a selection marker for parent stock lines to increase the growth performance of Amakusa Daioh cross chickens.

Effect of a Single Nucleotide Polymorphism in the Cholecystokinin Type A Receptor Gene on Growth Traits of the Miyazaki Jitokko Chicken.

The Miyazaki Jitokko chicken, native to the Miyazaki Prefecture in southern Kyushu Island, Japan, is the product of a three-way cross involving the Jitokko, White Plymouth Rock, and Kyushu Rhode breeds. In the present study, associations between a single nucleotide polymorphism (SNP; AB604331, g.420 C>A) of the chicken cholecystokinin type A receptor gene and growth traits in Miyazaki Jitokko chickens were investigated. Unrelated male birds (n=120) that had hatched on the same day were raised in the same chicken house and fed the same diet ad libitum from day 0 to 17 weeks of age. Body weight was recorded at 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, and 17 weeks and the average daily gain of each interval was calculated from the body weight data. SNP genotyping of each bird was performed using the mismatch amplification mutation assay. The associations between the SNP and growth traits were examined using the Thesias program. The genotype frequencies of AA, AC, and CC were 0.525, 0.383, and 0.092, respectively. AA birds were significantly heavier than CC birds from 4 to 17 weeks of age. In the estimated effect of alleles, body weight from 1 to 17 weeks of age in birds with the A allele was greater than that in birds with the C allele. During the rearing period, the effect of the A allele on average daily gain in the first half was greater than that in the second half. We conclude that the g.420 C>A SNP can be used as a selection marker for the parent stock lines of the Miyazaki Jitokko chickens to increase their growth performance.

Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens.

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3’ proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.

Phenotypic and Genotypic Examination of Antimicrobial Resistance in Thermophilic Campylobacter Species Isolated from Poultry in Turkey.

IntroductionThe study aimed to isolate thermophilic Campylobacter from chickens raised three rearing methods, determine its antimicrobial susceptibilities, and examine resistance-related genes by PCR.Material and MethodsCloacal swabs or intestinal contents were taken in Istanbul, Sakarya, and Izmir provinces. Chickens were from small village-based family-run businesses (n = 70), organically raised (n = 71), and conventionally raised broilers (n = 79). The samples were cultured on modified charcoal cefoperazone desoxycholate (mCCD) agar. Suspect isolates were identified with multiplex PCR (mPCR). As per EUCAST standards, MIC values were derived by broth microdilution for tetracycline, ciprofloxacin, nalidixic acid, kanamycin, gentamicin, and erythromycin in isolates of C. jejuni (n = 98) and C. coli (n = 83).ResultsIn C. jejuni, 78.6% tetracycline, 87.8% ciprofloxacin, and 81.6% nalidixic acid resistance was detected, but none was to kanamycin, gentamicin, or erythromycin. In C. coli, 98.8% ciprofloxacin and 63.9% nalidixic acid resistance was detected, whereas resistance to non-quinolones was not observed. C257T (Thr-86-Ile) mutation in the gyrA gene of all phenotypically quinolone-resistant isolates was detected through a mismatch amplification mutation assay PCR (MAMA-PCR). It emerged that all isolates bore the tet (O) resistance gene.ConclusionCommon tetracycline, nalidixic acid, and ciprofloxacin resistance exists in Campylobacter isolated from chickens raised three rearing methods.

Comparison of Mismatch Amplification Mutation Assay PCR and PCR-Restriction Fragment Length Polymorphism for Detection of Major Mutations in gyrA and parC of Escherichia coli Associated with Fluoroquinolone Resistance.

Fluoroquinolones are the drug of choice for most of the infections caused by Escherichia coli, and their indiscriminate use has resulted in increased selective pressure for antibiotic resistance. At present, sequencing is the only reliable and direct technique to detect mutations in the quinolone resistance determining region (QRDR). In this study, a rapid and reliable mismatch amplification mutation assay (MAMA) PCR to detect mutations in the QRDR was evaluated and compared to PCR-restriction fragment length polymorphism (PCR-RFLP). One hundred one clinical isolates of E. coli were subjected to MAMA-PCR and PCR-RFLP to detect QRDR mutations. Overall, 92 (91.08%) resistant isolates harbored a point mutation of S83L in gyrA. Double mutations in gyrA were also detected in 45 (44.55%) isolates. Similarly, 41 (40.59%) isolates possessed a point mutation at parC 80, and 25 (24.75%) isolates possessed a point mutation at parC 84. Additionally, MAMA-PCR-the first of its kind-was also standardized to detect mutations in regions gyrB 447 and parE 416, although no mutations were detected in these regions. The rapid and sensitive MAMA-PCR method evaluated in this study would be helpful in exploring the underlying mechanism of fluoroquinolone resistance to enhance control strategies.

Molecular detection ofserotype groups of Listeria monocytogenes isolated from gallbladder of cattle and sheep in Iraq.

AimThis study was designed to investigate the occurrence of serotypes of Listeria monocytogenes, an important food-borne pathogen, in gallbladder samples from cattle and sheep.Materials and MethodsThree hundred samples were collected and screened for the presence of L. monocytogenes. The identification of the isolates was confirmed by API-Listeria system and by the presence of hemolysin (hyl) gene. The isolates were subjected to polymerase chain reaction-based serotype identification with d1 (division 1), d2 (division 2), glt, mama (mismatch amplification mutation assay), and flaA (flagellin protein) genes.ResultsA total of 8 (2.7%) L. monocytogenes were recovered from 6 (4.0%) samples of sheep and 2 (1.3%) samples of cattle. All isolates showed positive results with Hly primers. Four isolates carried d1 gene, did not possess glt gene and harbored mama gene. The serotypes of these isolates were identified as 4a or 4c. The other 4 isolates carried d2 gene, 3 of them were positive with the FlaA primers, and hence, determined to be a 1/2a or 3a serotype, and 1 isolate was determined to be 1/2c or 3c serotype.ConclusionThis study concluded that the presence of 1/2a serotype in gallbladder samples indicates public health risk through cross-contamination of meat at slaughterhouses.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis.The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 102–105 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3–4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping.In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis.

Development and small-scale validation of a novel pigeon-associated mitochondrial DNA source tracking marker for the detection of fecal contamination in harvested rainwater

The current study was aimed at designing and validating (on a small-scale) a novel pigeon mitochondrial DNA (mtDNA) microbial source tracking (MST) marker for the detection of pigeon fecal matter in harvested rainwater. The pigeon mtDNA MST marker was designed to target the mtDNA Cytochrome b gene by employing mismatch amplification mutation assay kinetics. The pigeon marker was validated by screening 69 non-pigeon and 9 pigeon fecal samples. The host-sensitivity of the assay was determined as 1.00 while the host-specificity of the assay was 0.96. Harvested rainwater samples (n=60) were screened for the prevalence of the marker with the mtDNA Cytochrome b marker detected in 78% of the samples. Bayes' theorem was applied to calculate the conditional probability of the marker detecting true pigeon contamination and the marker subsequently displayed a 99% probability of detecting true pigeon contamination in the harvested rainwater samples. In addition, the mtDNA Cytochrome b marker displayed high concurrence frequencies versus heterotrophic bacteria (78.3%), E. coli (73.3%), total coliforms (71.1%) and fecal coliforms (66.7%). This study thus validates that targeting mtDNA for the design of source tracking markers may be a valuable tool to detect avian fecal contamination in environmental waters.

Polymorphisms in Lep and Lepr Genes in Infants: Correlation with Serum Leptin Values in the First 6 Months of Life

ABSTRACTObjective: Because several studies indicate that polymorphisms in leptin (Lep) and leptin receptor (Lepr) genes play a central role in determining obesity, we analyzed 2 single nucleotide polymorphisms (SNPs) in the Lep gene (Lep G2548A and A19G) and one in the Lepr gene (Lepr A668G) to verify the effect of the 3 SNPs on leptin concentrations in infancy.Methods: We enrolled 80 healthy Caucasian infants under 6 months of age, who were genotyped for the 3 SNPs with amplification refractory mutation system–mismatch amplification mutation assay (ARMS-MAMA) real-time polymerase chain reaction (PCR). Serum leptin values were measured with a radioimmunoassay method. Statistical significance was set at p < 0.05.Results: There were no significant differences between individually analyzed leptin polymorphisms Lep G2548A and A19G and serum leptin levels (p > 0.05). Because we found that Lep G2548A and A19G are in linkage disequilibrium on chromosome 7, we performed the haplotype analysis for Lep G2548A and Lep A19G. We obtained higher serum leptin levels in infants with the GG/GG haplotype (p < 0.05). Regarding receptor, we found higher leptin levels in GG-genotype infants for Lepr A668G (p < 0.001). Considering the 3 SNPs together, we found higher serum leptin values in GG/GG-GG infants (LepG2548A/A19G-Lepr A668G; p < 0.001).Conclusion: We obtained higher serum leptin levels in infants with the GG genotype for Lepr A668G, with haplotype GG/GG for Lep G2548A/A19G, and with GG/GG-GG (LepG2548A/A19G-Lepr A668G); thus, it seems that the genotype GG could be a protector against obesity development in infancy and adulthood. Moreover, these data confirm that not variations in the Lep gene as well as in the Lepr gene could play a role in weight gain. Further studies are needed to evaluate the role of genetics and the environment in a predisposition toward obesity later in life.

Retrospective genomic analysis of Vibrio cholerae O1 El Tor strains from different places in India reveals the presence of ctxB-7 allele found in Haitian isolates.

A total of 45 strains of Vibrio cholerae O1 isolated from 10 different places in India where they were associated with cases of cholera between the years 2007 and 2008 were examined by molecular methods. With the help of phenotypic and genotypic tests the strains were confirmed to be O1 El Tor biotype strains with classical ctxB gene. Polymerase chain reaction (PCR) analysis by double - mismatch amplification mutation assay PCR showed 16 of these strains carried the ctxB-7 allele reported in Haitian strains. Sequencing of the ctxB gene in all the 45 strains revealed that in 16 strains the histidine at the 20th amino acid position had been replaced by asparagine and this single nucleotide polymorphism did not affect cholera toxin production as revealed by beads enzyme-linked immunosorbent assay. This study shows that the new ctxB gene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed - field gel electrophoresis showed four distinct Not I digested profiles showing that multiple clones were causing cholera in 2007 and 2008.

Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains.

Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain.