Restriction Endonuclease Patterns Research Articles

First Report of 16SrII Group Related Phytoplasma Associated with Areca Palm Yellow Leaf Disease on Areca catechu in China.

The areca palm, Areca catechu L., family Arecaceae is an important herbal medicine which has potential for the treatment of parasitic diseases, digestive function disorders and depression (Peng et al. 2015). Yellow leaf disease (YLD), caused by phytoplasma, is a destructive disease of Areca catechu. In 1981, the YLD was first discovered in Tunchang, Hainan, China. According to the investigation in 2020, the occurrence area of YLD was 32 102.38 hm2 in Hainan, China, resulting in 50%-60% yield loss. Previous researchers based on 16S rDNA gene PCR amplification analysis showed that YLD in Hainan was caused by 16SrI group phytoplasma (Che et al. 2010). In August, 2022, yellow leaf symptoms were observed on middle and lower leaves of Areca catechu. Forty symptomatic plants and three asymptomatic samples were collected in Wenchang, Hainan, China (19°33'9″N, 110°48'5″E). Forty-three samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Phytoplasma universal primers named P1/P7 (Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) for 16Sr DNA and primers named fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997) for tuf genes were used for amplifying phytoplasma sequences from isolated DNA samples by nested PCR. No fragment was amplified in asymptomatic plants and four out of forty symptomatic samples could amplify target fragment. R16F2n/R16R2 amplicons (1 248 bp) and fTufu/rTufu amplicons (845 bp) from four symptomatic Areca catechu samples were sequenced in BGI (https://genomics.cn/). The 16Sr DNA GenBank accession numbers of four positive strains (named HNWC5, HNDZ1, HNDZ3 and HNDZ6) were OQ586072, OQ586085, OQ586086, OQ586087, respectively and the tuf GenBank accession numbers were OQ595209, OQ595210, OQ595211, OQ595212, respectively. Sequence alignment showed that the 16S rDNA and tuf sequence of HNDZ1, HNDZ3 and HNDZ6 were 100% consistent. 16S rDNA of HNWC5 was 99.96% consistent with HNDZ1 and tuf of HNWC5 was 98.31% consistent with HNDZ1. Interestingly, blast search based on 16S rDNA gene of HNWC5 showed 100% sequence identity with that of 16SrII group phytoplasma such as 'Eclipta prostrata' phytoplasma strain Ep1(MH144204.1), 'Aeschynomene americana' phytoplasma isolate AA1(MH231157.1) and 'Acacia confusa' witches'-broom phytoplasma isolate HK6(ON408364.1). Blast search based on tuf gene of HNWC5 showed 98.7% sequence identity with that of bamboo witches'-broom phytoplasma (FJ853160.1) and 91.02% sequence identity with that of 'podocarpus nagi' fasciation phytoplasma (KR633146) and 90.78% sequence identity with that of 'Musa acuminata' elephantiasis disease phytoplasma (MF983708). The phylogenetic tree was constructed based on 16Sr DNA gene by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al. 2016). The result indicated that the HNWC5, HNDZ1, HNDZ3 and HNDZ6 phytoplasma strains clustered a subclade in 16SrII group. The virtual RFLP analysis based on the 16Sr DNA gene sequence was performed by the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) using restriction endonucleases of AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI. The result indicated that HNWC5 was most similar to the reference pattern of peanut witches'-broom phytoplasma (16SrII-A subgroup, GenBank accession: L33765) and the pattern similarity coefficient of HNWC5 is 1.00. However, the HpaII restriction endonuclease pattern of HNDZ1, HNDZ3 and HNDZ6 was different from L33765 and the similarity coefficient was 0.97, which indicated this strain may represent a new subgroup within the 16SrII group. To our knowledge, this is the first report of 16SrII group related phytoplasma associated with YLD on Areca catechu in China. Our study contributes to understanding the polymorphism of phytoplasma causing YLD and provides an important reference for pathogen specific detection.

Isolation and characterization of three bacteriophages infecting Erwinia amylovora and their potential as biological control agent

BackgroundFire Blight, incited by Erwinia amylovora, is one of the most damaging pear and apple diseases in the world. Fire blight was introduced to Egypt in the 1960 and threatens the Egypt’s costs for pear industry. Currently, Phage therapy is considered to be secured biological method for controlling plant bacterial diseases. This investigation aimed to isolate and identify molecularly for bacteria causing fire bright disease. As well as isolation and identification bacteriophages via spot and plaque assay techniques from pear fire blight lesions and soil. On the other hand, bacteriophages were identified based on plaque morphology, virion morphology, physical characters, profile of DNA restriction and protein.ResultsPathogenicity test revealed that healthy seedlings and pear fruits were responsive to fire blight E. amylovora. Considering the relatively wide host range and greatest protein and genetic variability, using restriction enzyme pattern, the three diversity phage isolates named, EAP1, EAP2 and EAP3 showed a lack of diversity out of five were fatherly characterized. The phages confirmed the close relation of EAP1, EAP2 to Siphoviridae (hexagonal head and long flexible non-contractile tail) and EAP3 to Myoviridae (icosahedral head and contractile tail). The phages retained higher lytic competence of 90.4; 92.68 and 95.25% for EAP1, EAP2 and EAP3, respectively. The phages were stable at strong alkaline (pH 10) 2% salt solution conditions and UV spectra. While EAP3 phage revealed the hexagonal head and very short tail that belongs to Myoviridae family. Bacteriophages were characterized by digestion of the phage DNA with three restriction endonucleases and were placed into three groups based on the patterns. Bacteriophages were 9 used for reducing bacterial infection populations and severity on pear. In a bioassay, the biocontrol of E. amylovora was evaluated using disks of immature pear fruit. On the pear disk surface, bacterial exudate was considerably suppressed by all phage isolates. According to measurements of the bacterial population still present on the disk surface, phage therapy could reduce it by up to 97%. Bacteriophages reduced pear fire blight disease severity on pear fruit trails.ConclusionThe results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora. Bacteriophages reduced pear fire blight disease severity on pear fruit trials. The results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora.

The Prevalence and Molecular Distinguishing of Brucella melitensis Rev1 Strain among Field Isolates the Brucella from Sheep and Goat Milk through PCR-RFLP Analysis of omp2 Gene Polymorphism

The present study was undertaken to characterize the prevalence and molecular distinguishing of Brucella melitensis field and vaccine Rev1 strains isolated from milk among the sheep and goat population. The study was carried out on flocks of sheep and goats suffering from Brucella infection outbreaks. A total of 171 milk samples were collected from different districts of the West Bank, part of Palestine, and isolated on Brucella agar. 86 cultures were confirmed by standard biochemical methods and screened for Brucella melitensis 16M of IS711 element by a conventional PCR technique. All these samples were also amplified for the omp2 gene to identify differences between field and Rev 1 strains in an infected flock. The results obtained from the PstI restriction enzyme pattern revealed that only 2 (4.5%) samples have specific polymorphism accomplished with the Brucella melitensis Rev1 strain. All other samples were restricted according to the Brucella melitensis 16M polymorphism pattern and originated from nonvaccinated flocks. Despite the adverse side effects of the Brucella melitensis vaccine, only a well-organized whole-flock vaccination and awareness campaign may inhibit the virulence of the field strain and, subsequently, reduce the prevalence of brucellosis infection among animals and humans in Palestine.

Molecular analysis of acid-stable alpha-amylase (asAA) gene in Aspergillus niger using PCR-RFLP

Introduction and Aim: Amylase is an important enzyme with vast applications in various industries such as food and therapeutic industries. Aspergillus niger is commercially engaged in the making of alpha-amylase. Acid-stable alpha -amylase is mostly produced with microorganisms such as Bacillus and Aspergillus. The aim of this research was the molecular investigation of the acid-stable alpha-amylase (alpha-sAA) gene in A. niger. Materials and Methods: Sixty-three A. niger isolates were evaluated in this study. PCR method was performed for amplification of a 347 bp DNA band of the alpha-sAA gene. The Hpa II Restriction endonuclease was used for the digestion of PCR fragments. Results: A 347 bp DNA fragment was recovered from 49 out of 63 (78%) isolates. After cutting the PCR products with the HpalphaII enzyme, 81.6% of isolates showed the expected band and 18.4% presented different restriction endonuclease patterns. Conclusion: The results demonstrated the PCR-RFLP technique performed in this research was a valuable tool for analysis of the alpha-sAA gene in A. niger isolates.

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Toxoplasma gondii infection is a serious major public health concern. Toxoplasmosis is a disease that affects humans and warm-blooded animals all over the world. The virulence and severity of the sickness may be influenced by parasite load. The three clonal lineages for biological research are T. gondii genotypes I, II, and III. Using primer genes for gra1, rop1, and mic3, which have been identified as essential proteins for tachyzoite invasion and replication in host cells. PCR was used to determine the genotype of a T. gondii isolate from the Indonesian research center for veterinary science. In silico restriction site analysis was performed on T. gondii isolates using CLC sequence viewer 8.0 software to assess sequence data for the existence of restriction enzyme patterns. Predictable restriction fragment length polymorphism using In silico analysis. T. gondii sequencing genes are In silico digested with different restriction enzymes. The findings show that they can easily distinguish archetypal parasites from biotypes and estimate the genetic diversity of the parasites. According to the interpretation of the data, isolate T. gondii belongs to strain RH genotype I.

Unequal crossing-over: a common basis of single alpha-globin genes in Asians and American blacks with hemoglobin-H disease

The α-globin genes of five black Americans, two Chinese, and five Filipinos with HbH disease (an α-thalassemia state in which there is a single functional α gene) were analyzed by restriction endonuclease techniques. All subjects were found to have one chromosome 16, lacking both α genes, and another containing a single α gene ( –/-α). Restriction endonuclease patterns of the DNA obtained from all 12 subjects were identical and compatible with unequal crossing-over as the mechanism of origin of the single α gene in these individuals.

Isolation and characterization of bovine herpes virus 5 (BoHV5) from cattle in India.

Bovine herpesvirus 1 (BoHV1) and 5 (BoHV5) are genetically and antigenically related alphaherpesviruses. Infection with one virus induces protective immunity against the other. However, disease associated with BoHV1 and BoHV5 varies significantly; whereas BoHV1 infection is usually associated with rhinotracheitis and abortion, BoHV5 causes encephalitis in cattle. BoHV5 outbreaks are sporadic and mainly restricted to the South American countries. We report BoHV5 infection for the first time from aborted cattle in India. Based on the characteristic cytopathic effects in MDBK cells, amplification of the viral genome by PCR, differential PCR for BoHV1/BoHV5, nucleotide sequencing and restriction endonuclease patterns, identity of the virus was confirmed as BoHV5 subtype A. Serum samples from the aborted cattle strongly neutralized both BoHV1 and BoHV5 suggesting an active viral infection in the herd. Upon UL27, UL44 and UL54 gene-based sequence and phylogenetic analysis, the isolated virus clustered with BoHV5 strains and showed highest similarity with the Brazilian BoHV5 strains.

Analysis of Endonuclease activity in clinically and Environmental originated vibrio species.

The research under given depicts the study vibrio species taken from faecal and environmental source. It is well known through researches that environment possess a very dense presence of cholera causing vibrio strains and that only a certain subsets of the strains are associated with human disease. The difference in the genetic profile of the clinical strains of the Vibrio species compared with their environmental counterparts was here exploited to trace specific human isolates to vectors of disease and also to allow a better estimation of whether a particular genotype is more often associated with disease or not. Isolation of Vibrio species was performed by taking water sample from river and Faecal matter they were cultured on TCBS Agar plate further they were screened through their biochemical properties. 6 different isolates were chosen to sonicate and bring up the lysates in order to obtain to assay their endonuclease activity, In every nook and cranny endonuclease activity was possessed by each and every isolate1_FS were in 13 fragments ,2 _WS in 10 fragments ,3_FS in 11 fragments. Thus it gave comparative assessment of Restriction endonuclease pattern.

Molecular diversity of hpd gene in clinical isolates of Haemophilus influenzae

Infections due to Haemophilus influenzae result in tremendous global morbidity. The conjugated vaccines against H. influenzae type b (Hib) have dramatically reduced the incidence of invasive Hib disease in the routine immunization of infants. The several proteins used as vaccine candidates for this pathogen, but they don't produce efficient immune in animal models against all strains of H. influenzae. This study aimed to determine the diversity of hpd gene nucleotide sequences of Iranian native clinical isolates of H. influenzae as a native vaccine candidate compared to standard strains.Twenty isolates of H. influenzae recovered from different clinical specimens of patients admitted to Milad and Imam Khomeini hospitals, Tehran, Iran. Then, isolates detected and identified as H. influenzae using biochemical tests, and further confirmation through omp6 gene PCR. The hpd gene was amplified by PCR using gene-specific primers, and the amplicons digested with EcoR1. For four isolates, the Amplicon of hpd gene sequenced, and the sequences aligned with sequences harbored in GenBank. Subsequently, sequences were submitted to the EMBL site (http://www.ebi.ac.uk/embl/).EcoR1 restriction enzyme pattern was the same among the 19 clinical isolates, and only one isolate was different. That different one with 3 out of 19 isolates were sequenced. The results showed that the nucleotide sequences and the deduced amino acid sequences for protein D in clinical isolates were highly conserved with similarities >95%.In conclusion, regarding high similarity up to 99% in clinical isolates, protein D can be a novel vaccine candidate against all types of H. influenza from Iran. This finding should be proved with more isolates, and also, evaluate the immunological features of protein D in animal models.

From Fish Eggs to Fish Name: Caviar Species Discrimination by COIBar-RFLP, an Efficient Molecular Approach to Detect Fraud in the Caviar Trade.

The demand for caviar is growing as is its price on the market. Due to the decline of true caviar production from sturgeons, eggs from other fish species and other animals have been used as substitutes for caviar. The labels on these products should indicate the species from which the eggs were derived, but the label can be misleading in some cases. In this context, species identification using DNA analysis is crucial for traceability and authentication of caviar products. In this work, we applied the COIBar-RFLP procedure to obtain species-specific endonuclease restriction patterns useful to discriminate “caviar” species. The tested caviar products were identified as originating from eight species: Acipenser transmontanus, A. gueldenstaedtii, A. stellatus, A. baerii, Mallotus villosus, Huso huso, Cyclopterus lumpus and Eumicrotremus orbis. The results demonstrated that 14% of the caviar products examined have a label that does not indicate the species from which the eggs were originated. The MboI restriction enzyme produced specific profiles discriminating the eight species, confirming that the COIBar-RFLP is a useful approach for routine screening of seafood products due to its ease and rapid execution, as the results of screening can be obtained within 7 h, by-passing the need for sequencing.

Retraction Notice: Isolation and Characterization of an Aeromonas punctata Bacteriophage.

[This retracts the article on p. 185 in vol. 9.].

Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS11Q3 and BPS15Q2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS11Q3 and BPS15Q2 in food.

14. Engineering and Characterization of a Cloned Adenoviral-Library to Explore Natural Virus Diversity

Large double-stranded DNA (dsDNA) viruses such as adenovirus (Ad), herpesvirus, poxvirus and their recombinant counterparts were well explored in basic virology and biomedical research. Especially for the development of novel vaccines and gene therapies, Ad gained special attention and represents the most widely explored vector worldwide. Although ~70 types of human Ad and numerous nonhuman Ad (>200) have been identified so far a system for efficient Ad genome cloning and manipulation was lacking and therefore the majority of recombinant adenoviral vectors (AdVs) are based on a small fraction of Ad types. Here we report the generation and characterization of an engineered human adenoviral-library allowing exploration and system studies of the natural Ad diversity. Towards that end we first established that adenoviral genomes can be cloned and tagged in a high-throughput manner utilizing advanced homologous recombination techniques. Wild type Ads from clinical isolates including around half of the currently known adenovirus types that represent all seven human adenovirus species were propagated and direct high-throughput cloning (HTC) applied. The integrity of cloned Ad genomes was confirmed by DNA restriction enzyme pattern and virus reconstitution was conducted using optimized conditions. Most importantly next-generation sequencing (NGS) and phylogenetic analysis was performed. As a further step, half of all cloned adenoviruses representing each species were tagged with a 2A peptide-mediated multicistronic expression cassette providing a Turbo Green fluorescent protein as in vitro marker, a NanoLuc luciferase for in vivo studies and kanamycin/neomycin as selection marker. For HTC of tagged viruses the reporter cassette was inserted into the adenovirus E3 region using different orientations because the orientation of the transgene was essential for reconstitution efficiencies. After successful reconstitution these double reporters-labeled AdVs were evaluated in cultured cell lines and mouse models. In vitro characterization revealed distinct tropisms for tested viruses. Among the currently evaluated cell lines, specie B viruses demonstrated high transduction efficiencies in epithelial (Hela- and A549 cells) and endothelial cells, while Ad5 still displayed highest transduction rates in other human and murine cell types (hepatocytes, lymphocytes, neuroblastoma cells and myoblasts). For the further characterization of unknown receptor usage, we injected recombinant viruses into DSG2 or CD46 transgenic mice for in vivo bio-distribution analyses on genome level by quantitative PCR and on protein level by immunohistology analyses. We anticipate that our engineered adenoviral-library will provide a spacious novel view to the adenovirus field. As a broader perspective it will bring AdV and also other large dsDNA viruses from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology.

Identification of clinical isolates of Acinetobacter baumannii from Iran and study of their heterogeneity

BackgroundAcinetobacter baumannii has become one of the most serious causative agents of nosocomial infections due to its significant ability to survive on hospital surfaces. It is mainly an emerging opportunistic pathogen infecting patients in intensive care units. This study was aimed to identify the clinical isolates of A. baumannii and to investigate their heterogeneity using polymerase chain reaction (PCR)-based typing methods. MethodsA total of 197 nonduplicate isolates recovered from a wide range of clinical samples were subjected to conventional cultural and biochemical tests. For those isolates that were preliminary identified as A. baumannii, rpoB-based PCR with subsequent restriction fragment length polymorphism (RFLP) using two restriction enzymes (TagI and HaeIII) was performed to investigate the genetic diversity of the strains and their presumptive relationships with different clinical presentation of the disease caused by this pathogen. ResultsIn total, 50 isolates (25.4%) were identified as A. baumannii using conventional phenotypic methods with subsequent confirmation by rpoB sequencing. RFLP analysis demonstrated five different restriction enzyme patterns, designated as A–E clusters. Most A. baumannii isolates were categorized under Cluster A (32%). We found no significant relationship between clinical presentation and the clustering of the isolates. ConclusionThis study showed that the rpoB region possesses high discriminatory power to identify the isolates to the species level. This marker showed high interspecies variability that might be useful for strain typing. The results also suggest the possibility of the existence of a predominant clone of A. baumannii among infected patients in Iran.

Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism.

The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species.

Isolation and Characterization of an Aeromonas punctata Bacteriophage.

An Aeromonas punctata bacteriophage, named as DH1, was isolated from East Lake, Wuhan city, China. Morphologically, phage DH1 showed a typical Myoviridae structure consisting of an isometric head (50 nm in diameter) and a visible tail. The bacteriophage had a latent period of about 90 minutes and an average burst size of about 125 PFU•Cell-1. Restriction enzyme pattern of the bacteriophage’s genome showed that the genome is a double-stranded DNA and about 34kb in size. The sequenced genomic fragments showed highly similarities to gp04 and gp16 sequence of other Myoviridae bacteriophages at protein level.

Identificación de especies de Meloidogyne asociadas a plantas ornamentales de altura en Costa Rica.

<p>El objetivo del presente trabajo fue identificar especies de nematodos del género <em>Meloidogyne </em>asociadas a plantas ornamentales de altura. El estudio se realizó en el cantón de San Isidro de Heredia, Costa Rica, en un vivero comercial durante el periodo 2011-2012, en diez especies de plantas. Para la identificación se utilizaron mediciones morfométricas de la longitud del estilete, tamaño de cola y región hialina de los juveniles en segundo estado de desarrollo (J2); también se obtuvieron diseños perineales de hembras ovígeras. Se extrajo ADN genómico de un solo individuo J2 y se realizaron análisis mediante la amplificación del espacio intergénico entre la subunidad II del citocromo oxidasa (COII) y la subunidad larga del gen ARN ribosomal mediante la técnica PCR-RFLP. La integración de estos métodos permitió identificar cinco especies de nematodos del género <em>Meloidogyne </em>(<em>M. arenaria</em>, <em>M. hapla</em>, <em>M. hispanica, M. incognita</em>, <em>M. javanica</em>), y se reportaron nuevos patrones de restricción con la enzima AluI para <em>M. hapla </em>y <em>M. javanica</em>. Además se obtuvo el reporte preliminar de <em>M. hispanica</em>, descrito mediante la secuenciación de las regiones del 18S y 28S.</p>

Genetic homogeneity among Leishmania (Leishmania) infantum isolates from dog and human samples in Belo Horizonte Metropolitan Area (BHMA), Minas Gerais, Brazil.

BackgroundCertain municipalities in the Belo Horizonte Metropolitan Area (BHMA), Minas Gerais, Brazil, have the highest human visceral leishmaniasis (VL) mortality rates in the country and also demonstrate high canine seropositivity. In Brazil, the etiologic agent of VL is Leishmania (Leishmania) infantum. The aim of this study was to evaluate the intraspecific genetic variability of parasites from humans and from dogs with different clinical forms of VL in five municipalities of BHMA using PCR-RFLP and two target genes: kinetoplast DNA (kDNA) and gp63.MethodsIn total, 45 samples of DNA extracted from clinical samples (n = 35) or L. infantum culture (n = 10) were evaluated. These samples originated from three groups: adults (with or without Leishmania/HIV co-infection; n = 14), children (n = 18) and dogs (n = 13). The samples were amplified for the kDNA target using the MC1 and MC2 primers (447 bp), while the Sg1 and Sg2 (1330 bp) primers were used for the gp63 glycoprotein target gene.ResultsThe restriction enzyme patterns of all the samples tested were monomorphic.ConclusionsThese findings reveal a high degree of genetic homogeneity for the evaluated gene targets among L. infantum samples isolated from different hosts and representing different clinical forms of VL in the municipalities of BHMA studied.

Identification of four Donax species by PCR–RFLP analysis of cytochrome c oxidase subunit I (COI)

Four Donax species, D. semistriatus, D. trunculus, D. variegatus and D. vittatus, are found on European coasts. Nevertheless, despite their economic importance there is not a reliable method to differentiate these species independently of their size or condition. Such a method could help to protect consumer rights and avoid commercial fraud due to the replacement of valuable species by less valuable ones with similar morphological traits. In this work, the sequence of the mitochondrial cytochrome oxidase subunit I region was examined in individuals of these species to identify restriction site variation and develop polymerase chain reaction–restriction fragment length polymorphisms assays. Species-specific restriction endonuclease patterns were found with the enzymes AluI, HaeIII and MspI, allowing an exact identification of Donax species. This methodology provides simple, reliable and cost-effective identification of four Donax species and may be useful to prevent commercial fraud and to increase food traceability.

Salmonellosis and Meat Purchased at Live-Bird and Animal-Slaughter Markets, United States, 2007–2012

Salmonellosis and Meat Purchased at Live-Bird and Animal-Slaughter Markets, United States, 2007–2012