Random Amplification Of Polymorphic DNA-polymerase Chain Reaction Research Articles

Antibiotic resistance and phylogenetic comparison of human, pet animals and raw milk Staphylococcus aureus isolates

The present study was conducted to compare the S. aureus isolates from different sources in the basis of resistance phenotypic and genotypic features and phylogenetic differences. Total of 70 S. aureus isolates (including 25 human, 25 raw milk and 20 pet animal isolates) were subjected to the antimicrobial susceptibility testing, polymerase chain reaction (PCR) detection of the resistance genes and DNA fingerprinting using random amplification of polymorphic DNA–PCR (RAPD-PCR) to survey the variability of the isolates. Among 70 S. aureus, 55 (78.5%) isolates were MRSA. The isolates showed the highest antibiotic resistance to methicillin, ampicillin and penicillin (78.5%) and showed the lowest resistance to ciprofloxacin (12.8%). ErmB and tetM resistance genes were present in all isolates and the vanA gene was not detected in any of the isolates. Thirteen distinct clusters were identified in RAPD-PCR fingerprinting. Statistical analysis showed that the isolates without resistance to antibiotics were significantly in associated with raw milk origin (P < 0.05). According to the results of the study, S. aureus strains with pets and raw milk origin are significant sources of antibiotic-resistant isolates such as MRSA. They are also carriers of resistance genes that can be transmit to human isolates and cause drug resistance in human infections. Identifying the source of these infections is possible with a reliable genotyping method such as RAPD-PCR.

Insecticidal and Genotoxic effects of some indigenous plant extracts in Culex quinquefasciatus Say Mosquitoes

Five different weed plants viz. Convulvulus arvensis, Chenopodium murale, Tribulus terrestris, Trianthema portulacastrum, and Achyranthes aspera were investigated for their entomocidal and genotoxic effects against Culex quinquefasciatus mosquitoes. High mortality was observed at 72 hours in a dose dependent manner. Among all the tested plants, A. aspera was found highly significant which showed 100% mortality at 250 ppm after 72 hours with LC50 of 87.46, 39.08 and 9.22 ppm at 24, 48, respectively. In combination with Bacillus thuringiensis israelensis (Bti); A. aspera also caused 100% mortality at 250 ppm concentration after 72 hours (LC50 8.29 ppm). Phytochemical analysis of all the tested weed plants showed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. Random Amplification of Polymorphic DNA-Polymerase chain reaction (RAPD-PCR) and comet assay were performed to assess the genotoxic effect of A. aspera but no change in DNA profile was observed. Furthermore, FTIR showed the presence of phenolic compounds in A. aspera extract. It is suggested that certain phenolic compounds such as flavonoids modulate the enzymatic activity and, hence, cause the death of larvae of Cx. quinquefasciatus. Altogether, current study would serve as an initial step towards replacement of synthetic insecticides to plant-microbe based biopesticide against Culex mosquitoes in future.

In vitro assessment of Schistosoma mansoni cercaricidal activities of Solanum nigrum and Callistemon citrinius leaves extracts and cercarial genetic changes by RAPD-PCR

Abstract: Background: Schistosomiasis is contracted by exposure to fresh water containing cercariae that develop into adult worms after penetration of human skin. Interruption of schistosomiasis vital cycle by elimination of cercariae may enhance methods of transmission control. Objective: To evaluate the vitro effect of methanol extracts of Solanum nigrum ( S.nigrum) and callistemon citrinus (C. citrinus) leaves on Schistosoma mansoni cercarial genetic makeup by random amplification of polymorphic DNA-Polymerase chain reaction (RAPD-PCR). Material and methods: The effect different concentrations of both plants extracts on cercarial morphology and mortality was observed with different lethal concentrations (LC). Also, assessment of DNA change in exposed S.mansoni cercariae to LC50 of both plants in comparsion with non-exposed ones by RAPD-PCR assay was investigated. Cercariae were divided into three groups: group A: control non-exposed cercariae; group B: cercariae exposed to S.nigrum; and group C: cercariae exposed to C.citrinus. Results: The cercaricidal potency of tested extracts was concentration-dependent. The cercaricidal toxicity of S. nigrum extracts was 1.2 times higher than that of C.citrinus(LC90 values were 50 mg/L and 60 mg/L, respectively). RAPD-PCR revealed different band polymorphism patterns for each primer used and cercariae exposed to S.nigrum revealed a higher number of band polymorphism (20 bands) than that obtained by cercariae exposed to C.citrinus (16 bands) which were different from those of control group reflecting the genetic variability among the groups studies. Conclusion: C.citrinus and S.nigrum are effective cercaricidal agents that can be utilized to minimize water transmission of schistosomiasis. Also, RAPD-PCR is useful for examining the genetic polymorphism of schistosomal cercariae induced by plants extracts, and assessment of genetic of damage of drug development fields.

An Outbreak of ESBL-producing Klebsiella pneumoniae in an Iranian Referral Hospital: Epidemiology and Molecular Typing.

Klebsiella pneumoniae is a common cause of nosocomial infections; however, there is limited information in Iran regarding nosocomial outbreaks due to extended-spectrum β-lactamase (ESBL) producing K pneumoniae strains, particularly using molecular methods. The present study focused on the molecular mechanism of ESBL resistance and genetic relatedness in K. pneumoniae isolates causing nosocomial infections in an Iranian referral hospital. This study evaluated the antimicrobial resistance and molecular epidemiology of K. pneumoniae causing nosocomial infections in children between October 2013 and March 2014. The ESBL detection was carried out for all the isolates by the CLSI method and PCR was carried out for the detection of the blaSHV, blaTEM, and blaCTX-M genes among ESBL-producing K. pneumonia. Molecular typing of the K. pneumoniae was performed using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total of 30 isolates of K. pneumoniae were used for epidemiological analysis. High rates of resistance to cefotaxime (n=29, 97%), cefazolin (n=29, 97%), cefepime (n=25, 83%) and gentamicin (n=23, 77%) were observed. A total of 29 strains (97%) produced ESBLs. The frequency of blaSHV, blaCTX-M and blaTEM genes among these isolates was 83% (n=25), 70% (n=21) and 57% (n=17), respectively. Surprisingly 11 isolated (37%) carried blaSHV, blaCTX-M and blaTEM genes simultaneously. Moreover, the concurrent presence of "blaSHV and blaCTX-M" and "blaSHV and blaTEM" was seen in 8 (27%) and 4 (13%) isolates, respectively. RAPDPCR analyses revealed that K. pneumoniae isolates belonged to 2 RAPD-PCR types among which one cluster counted for 28 isolates. To our knowledge, this is the first published report of a nosocomial outbreak of ESBL-producing K. pneumoniae in children in Iran. Although the epidemiology of nosocomial infections with ESBL-producing organisms has not yet been explored in depth in Iran, our findings suggest that ESBL-producing organisms are already an established public health threat in our country.

Diversity and metallo-β-lactamase-producing genes in Pseudomonas aeruginosa strains isolated from filters of household water treatment systems

The microbiological quality of drinking water has long been a critical element in public health. Considering the high clinical relevance of Pseudomonas aeruginosa, we examined the filters of household water treatment systems for its presence and characteristics to determine the systems' efficiency in eliminating the bacteria. In total, filters of 50 household water treatment systems were examined. Microbiological and molecular methods were used for the detection and confirmation of P. aeruginosa isolates. Random Amplification of Polymorphic DNA-polymerase chain reaction (RAPD-PCR) was performed to detect similarities and differences among P. aeruginosa isolates. Combined disk (CD) method and double disk synergy test (DDST) were performed to detect metallo-beta-lactamase (MBL)-producing P. aeruginosa isolates. Finally, PCR was performed to detect MBL genes in MBL-producing strains. From the 50 analyzed systems, 76 colonies of P. aeruginosa were identified. In some systems, isolated bacteria from different filters harbored similar genetic profiles, indicating that these isolates may be able to pass through the filter and reach higher filters of the system. Phenotypic tests revealed 7 (9.2%) MBL-producing strains. Two isolates were positive for blaVIM-1, whereas one isolate was positive for blaNDM and blaIMP-1. The wide distribution of resistant phenotypes and genetic plasticity of these bacteria in household water treatment systems indicate that resistance mechanisms circulate among P. aeruginosa isolates in the environment of the filtration systems. The presence of MBL-producing genes in these systems and P. aeruginosa as a potential reservoir of these resistance genes can be a major concern for public health.

The effect of psychrotrophic bacteria isolated from the root zone of winter wheat on selected biotic and abiotic factors

Abstract The roots of winter wheat plants, cv. Mikon, grown in 45-year monoculture, were analysed in the study. Twenty-two bacterial isolates obtained from the rhizosphere, rhizoplane, and endorhizosphere that were capable of growth at 8°C and at 28°C, were selected for further analysis. The isolated psychrotrophs accounted for 25% of all bacteria present in the wheat rhizosphere and capable of growth at 8°C. Psychrotrophic bacteria were analysed at a temperature of 10°C and 28°C to determine their ability to inhibit the growth of pathogenic fungi, solubilise mineral phosphates, and to determine their ability to degrade chitin and cellulose. Similarity between the isolates was determined by Enterobacterial Repetitive Intergenic Consensus – Polymerase Chain Reaction (ERIC-PCR) and Random Amplification of Polymorphic DNA – Polymerase Chain Reaction (RAPD-PCR). The majority of isolated psychrotrophs inhibited the growth of pathogenic fungi and solubilised mineral phosphates at both incubation temperatures. Psychrotrophic bacteria exerted a two-fold stronger inhibitory effect on mycelial growth at 10°C than at 28°C. The growth of Fusarium culmorum and F. oxysporum was inhibited to the highest extent at 10°C and at 28°C, respectively. Phosphate solubilisation rates were higher at 28°C, particularly in the rhizosphere. Regardless of temperature, the bacteria exhibited low chitin-degrading potential, and none of the isolates was capable of degrading cellulose. A high similarity between the selected psychrotrophs was revealed by ERIC-PCR and RAPD- -PCR analyses. Based on RAPD-PCR, the analysed population was divided into a group of isolates obtained from the rhizosphere, and two groups comprising representatives of both the rhizoplane and the endorhizosphere. Due to their ability to grow over a wide temperature range and increase phosphorus availability to plants, and their antagonism against pathogens, psychrotrophic bacteria can be used to improve the growth and yield of cereal crops.

The role of the immunological background of mice in the genetic variability of Schistosoma mansoni as detected by random amplification of polymorphic DNA.

Schistosomiasis is a parasitic disease caused by flatworms of the genus Schistosoma. Among the Schistosoma species known to infect humans, S. mansoni is the most frequent cause of intestinal schistosomiasis in sub-Saharan Africa and South America: the World Health Organization estimates that about 200,000 deaths per year result from schistosomiasis in sub-Saharan Africa alone. The Schistosoma life cycle requires two different hosts: a snail as intermediate host and a mammal as definitive host. People become infected when they come into contact with water contaminated with free-living larvae (e.g. when swimming, fishing, washing). Although S. mansoni has mechanisms for escaping the host immune system, only a minority of infecting larvae develop into adults, suggesting that strain selection occurs at the host level. To test this hypothesis, we compared the Belo Horizonte (BH) strain of S. mansoni recovered from definitive hosts with different immunological backgrounds using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). Schistosoma mansoni DNA profiles of worms obtained from wild-type (CD1 and C57BL/6J) and mutant (Jα18- / - and TGFβRIIdn) mice were analysed. Four primers produced polymorphic profiles, which can therefore potentially be used as reference biomarkers. All male worms were genetically distinct from females isolated from the same host, with female worms showing more specific fragments than males. Of the four host-derived schistosome populations, female and male adults recovered from TGFβRIIdn mice showed RAPD-PCR profiles that were most similar to each other. Altogether, these data indicate that host immunological backgrounds can influence the genetic diversity of parasite populations.

Effects of garlic on Schistosoma mansoni harbored in albino mice: Molecular characterization of the host and parasite

Garlic has been used for its health benefits for thousands of years. Modern research confirmed many of the healing properties of garlic, including its antiparasitic activity. This study was designed to evaluate the antischistosomal action of garlic through detecting the changes in DNA profile of Schistosoma mansoni worms and the infected mouse. Forty mice were subcutaneously infected with ~200 Schistosoma mansoni cercariae/mouse. Infected mice were divided into four equal groups: non-treated, prophylactic, therapeutic, and continuously-treated. Non-infected control and garlic-treated groups were assigned for the sake of comparison. Garlic extract (50mg/kg bw/mouse) was given orally, day after day, at a fixed daytime. Seven weeks post-infection, adult schistosomes were recovered by perfusion and the livers of the mice were excised out and were processed for DNA extraction and Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The results showed that garlic exerted no major changes in the genome of schistosomes. Nevertheless, that schistosomal infection induced genetic alterations in the DNA of mice, and garlic was able to ameliorate such alterations to a great extent.

MOLECULAR CHARACTERIZATION OF GOATS USING RANDOM AMPLIFIED POLYMORPHIC DNA

Black Bengal goat is a very valuable goat breed in India. The present investigation was proposed to be undertaken to evaluate gene flow, phylogeographic history, genetic structure and differentiation of both the goat populations to study genetic variation prevail ing among Black Bengal and Black Bengal type goat in west Bengal and Jharkhand respectively and to study genetic relationship between both goat populations and to study population structure and gene flow between th ese two goat populations. This experiment was conducted in the department of animal breeding and genetics ( molecular biology laboratory). DNA was extracted from 50 goats from each of the Black Bengal and Black Bengal type goats of Jharkhand and the extracted DNA was observed by gel electrophoresis. Ten specific goat specific primers were obtained from sigma. All the primers except for primers one and two were capable of priming polymorphic amplification pattern in both the b reeds. Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 50 Black Bengal goat and 50 Black Bengal type goats of Jharkhand (referred to as Jharkhand black) goat breeds. Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. The amplific ation pattern of representative samples of Black Be ngal showed that primers 6,7,8,9 were highly polymorphic. Polymorphism Information Content (PIC) or expected heterozygosity scores varied from a minimum of 0.219 for primer sigma 06-3 to a maximum of 0.486 for primer sigma 04 with over all mean 0.411 in Black Bengal goat. Similarly for Jharkhand Black, the valu e of PIC ranged from a minimum of 0. 278 for primer sigma 06-3-0.496 for primer sigma10-2 with over all mea n value of 0.413. Data were analyzed by using a computer programe POPGENE (Version 1.31). A more appropriate measure of genetic variation within a p opulation is gene diversity .It was observed that, mean value of Nei,s Gene diversity value (h) was 0.3750 for Black Bengal and was 0.4022 for Jharkhand black. In case of Black Bengal, it was found that the maximum value of Nei’s gene diversity was 0.4861 for locus sigma 6-2 and sigma 9-2 while the minimum value of 0.0664 was found for locus Sigma 04. In Jharkhand Black t he maximum value of Nei’s gene diversity (h) was found to be 0.4965 for locus Sigma 10-2 and sigma06-1and the minimum value was 0.2491 for locus sigma 08-1. Dendrogram based on Nei’s genetic distance using Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated relative closeness and distance of the two goat breed. This study was mainly done on b reeds raised in marginal agricultural areas in orde r to assist in situ conservation. The result was importa nt with respect to puzzle of goat genetic diversity and conservation. Therefore it can be recommended that within breed diversity is actively maintained to en able these extensively unmanaged stocks to adapt to futu re demands and conditions and there is ample scope for further improvement in its productivity through app ropriate breeding strategies.

Molecular and Biochemical Studies on the Effect of Gamma Rays on Lead Toxicity in Cowpea (Vigna sinensis) Plants

The effect of lead acetate in the presence or absence of cowpea seeds irradiated with gamma rays on morphological criteria, protein electrophoresis, isozymes, and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) of leaves was investigated. A highly significant decrease in shoot and root length was observed upon lead acetate exposure (300 and 600μM). On the other hand, in seeds irradiated with gamma rays (2, 5, and 8krad), these morphological parameters were increased after lead acetate treatments. Meanwhile, all treatments (lead acetate and gamma rays) caused variations in number, intensity, and/or density of SDS electrophoretic bands of proteins. In addition, electrophoretic studies of esterase, acid phosphatase, peroxidase, polyphenol oxidase, catalase, and superoxide dismutase isozyme activities were increased with increasing the concentrations of lead acetate and gamma ray doses. The variation in DNA profile in response to lead acetate and gamma irradiation treatments was detected by RAPD-PCR technique. The result of RAPD analysis using the five primers indicated the appearance and disappearance of DNA polymorphic bands at all treatments (gamma rays and lead stress). The relatively high concentrations of lead acetate (600μM) induced more changes in genomic DNA pattern.

Re-identification of Aspergillus fumigatus sensu lato based on a new concept of species delimitation

The species concept of Aspergillus fumigatus sensu stricto has recently been defined by polyphasic taxonomy. Based on the new concept of species delimitations, 146 worldwide strains of Aspergillus fumigatus sensu lato were re-identified. Of those 146 strains, 140 (95.8%) could be identified as A. fumigatus sensu stricto, 3 (2.1%) as A. lentulus, and the remaining 3 strains as A. viridinutans complex, Neosartorya udagawae, and N. cf. nishimurae. Of 98 clinical strains, only 1 from dolphin nostril was identified as A. lentulus and not A. fumigatus sensu stricto. Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers PELF and URP1F produced nearly the same band patterns among 136 strains of A. fumigatus sensu stricto while discriminated the species from its related species. We also discussed about identification of several atypical A. fumigatus strains from clinical environments.

Molecular characterisation of UV and chemically induced mutants of Trichoderma reesei FCBP-364

Cellulases are a highly diverse group of enzymes whose function is crucial to the healthy functioning of the biosphere, since more than half of all biomass on the planet consists of their substrate, cellulose. Trials were conducted to study the effect of mutagenesis by ultraviolet (UV) irradiation (5–40 min) and ethyl methane sulphonate (EMS) treatment (50–300 µg mL−1) to obtain hyperactive cellulase enzyme producers. Putative mutants of Trichoderma reesei FCBP-364 were selected on the basis of their bigger hydrolysing zone formation and compared to the parental strains quantitatively. UV- and EMS-treated putative mutants of the test strains exhibited a 1.5–2-fold enhancement in enzymatic activity over the parental strain. The profile of genetic variability among native and mutant derivatives was scrutinised through random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). The scanned amplicons confirmed the modification in genetic make up which might be the cause of the stir up in the enzyme activity.

Characterization of Saprolegnia isolates from Persian sturgeon ( Acipencer persicus) eggs based on physiological and molecular data

Summary Objective To investigate the physiological characteristics and genetic variations of Saprolegnia species isolated from Persian sturgeon (Acipencer persicus) eggs. Materials and methods One hundred and fifty fish eggs covered with fungal hyphae were collected from propagation center of sturgeon and teleost fish Shahid Rajaii in Mazandaran province between March and May 2007. The samples were cultured into the specific media and the colony hyphal radial growth rates were determined at 18 °C and 12 °C. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for genetic variations of the isolates. Results All samples were positive for Saprolegnia species. Most fungal isolates grew well at 18 °C but no isolates grew at 12 °C. Repeated zoospore emergence was found in isolates of Saprolegnia species, which were genetically linked to reference S. parasitica. This phenomenon was not seen in isolates that genetically liked to reference S. diclina. The RAPD analysis among Saprolegnia isolates was counteracted from 385 amplified products in 21 separable positions and indicated that the fungal isolates were composed of four genetically distant groups. Conclusion We found different physiological characteristics and genetic variations of Saprolegnia isolates from Acipencer persicus eggs. Although repeated zoospore emergence was clearly not specific for pathogenic Saprolegnia species, it was present in certain groups of isolates defined by genotype and can be useful in distinguishing subgroups of pathogenic Saprolegnia species.

Molecular and probiotic characterization of bacteriocin-producing Enterococcus faecium strains isolated from nonfermented animal foods

The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as probiotics in raw and processed foodstuffs. 16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3.0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents. In our study, DNA sequencing of the 16S rRNA gene and RAPD-PCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract. This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.

Genetic Diversity of Red Chittagong Cattle Using Randomly Amplified Polymorphic DNA Markers

Problem statement: Red Chittagong Cattle (RCC) is a potential genetic resource in Bangladesh having dairy potentialities, high resistance to disease and survive harse environmental condition while require low input supply. This resource is now disappearing due to indiscriminate crossing with other local and exotic cattle. To conserve this resource, find out the genetic diversity of RCC is prime concern. Approach: To determine the genetic variation of Red Chittagong Cattle and to find out the genetic present status for their future improvement and conservation program. The experiment was conducted in the Molecular Genetics Laboratory under Animal Production Research Division, Bangladesh Livestock Research Institute (BLRI), Saver, Dhaka, Bangladesh. DNA was extracted from 100 RCC blood samples and the extracted DNA was observed by gel electrophoresis. Randomly 16 primers were tested among them three primers were matched and found polymorphic. Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of RCC. Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. Data were analyzed by using a computer program POPGENE (Version 1.31). Results: The highest and the lowest level of gene frequency value (0.9590 and 0.0440) were observed in allele 0 and 1 with BMC1222-2 locus and only one monomorphic locus (OPB07-1) was found. The Nei’s gene diversity and Shannon’s information indicated that the highest and the lowest diversity were found in Anwara (0.2925) and Chandonish (0.2147) respectively. The pair-wise mean genetic distance value ranged (below diagonal) from 0.0092-0.0279. The minimum distance was found between Satkania and Chandonish. On the other hand maximum distance was found between RCC BLRI and Potia. The dendrogram segregated the five population of RCC into two clusters: RCC BLRI and Anwara is one cluster whereas Satkania, Chandonish and Potia made another cluster. Conclusion: The genetic diversity of RCC is relatively higher for a prescribed breed and therefore, have an opportunities to improve them using selective breeding like ONBS.

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: Morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8–16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.

Molecular Characterization of Black Bengal and Jamuna Pari Goat Breeds By RAPD Markers

This experiment was conducted in the Genetics, Breeding and Reproductive Biotechnology Laboratory under Goat and Sheep Production Research Division, Bangladesh Livestock Research Institute (BLRI), Saver, Dhaka, Bangladesh. DNA was extracted from 14 goat breeds and the extracted DNA was observed by gel electrophoresis. Eight goat specific primers were synthesized by ASM-800 DNA synthesizer and screened in the study and all these primers were capable of priming polymorphic amplification pattern in both the breeds. Random amplification of polymorphic DNA - Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 14 black bengal goat and Jamuna pari goat breeds. Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. Data were analyzed by using a computer programe POPGENE (Version 1.31). Highest level of Nei's (1) gene diversity value (0.4898) was observed in BMS 1494 locus and the mean genetic diversity was obtained 0.3724 among the 14 goat breeds. The highest number of polymorphism obserbed in primer BM1818.The pair-wise genetic distance value ranged from 0.2500 to 1.000. Dendrogram based on Nei's (1) genetic distance using Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated segregation of the 14 goat breeds. Within Jamuna pari goat genetic similarity is low as well as black Bengal goat.

Molecular Phylogeny of Neotropical &lt;I&gt;Anopheles&lt;/I&gt; (&lt;I&gt;Nyssorhynchus&lt;/I&gt;) &lt;I&gt;albitarsis&lt;/I&gt; Species Complex (Diptera: Culicidae)

A phylogeny was reconstructed for four species belonging to the Neotropical Anopheles (Nyssorhynchus) albitarsis complex using partial sequences from the mitochondrial cytochrome oxidase I (COI) and NADH dehydrogenase 4 (ND4) genes and the ribosomal DNA ITS2 and D2 expansion region of the 28S subunit. The basis for initial characterization of each member of the complex was by correlated random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Analyses were carried out with and without an outgroup (An.(Nys.) argyritarsis Robineau-Desvoidy) by using maximum parsimony, maximum likelihood, and Bayesian methods. A total evidence approach without the outgroup, using separate models for "fast" (COI and ND4 position 3) and "slow" (rDNA ITS2 and D2, and COI and ND4 position 1) partitions, gave the best supported topology, showing close relationships of An. albitarsis Lynch-Arribálzaga to An. albitarsis B and An. marajoara Galvão & Damasceno to An. deaneorum Rosa-Freitas. Analyses with the outgroup included showed poorer support, possibly because of a long branch attraction effect caused by a divergent outgroup, which caused one of the An. marajoara specimens to cluster with An. deaneorum in some analyses. The relationship of the above-mentioned result to a separately proposed hypothesis suggesting a fifth species in the complex is discussed.

Molecular Characterization of Rathi and Tharparkar Indigenous Cattle (Bos indicus) Breeds by RAPD-PCR

Random amplification of polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 30 animals of Rathi cattle and 42 animals of Tharparkar cattle. Genomic DNA was isolated as per standard protocol and evaluated for its quality, purity and concentration. Twenty three random primers were screened out of which 15 primers yielded satisfactory amplifications and were used for further analysis. Average numbers of polymorphic fragments per primer were 7.07±0.86 in Rathi and 6.80±0.61 in Tharparkar cattle. The percentage of polymorphic bands in these two cattle breeds were 86 and 87%, respectively. Within breed genetic similarities for pooled over primers in the animals of Rathi and Tharparkar breeds were 0.577±0.30 and 0.531±0.02, respectively on the basis of band frequency (BF) and 0.645±0.04 and 0.534±0.04, respectively on the basis of band sharing (BS). Averages of between breed genetic similarities for pooled over primers were 0.97 and 0.92 according to BF and BS, respectively, which reflect higher degree of genetic similarity between Rathi and Tharparkar cattle breeds. Index of genetic distance based on BF and BS for pooled over primers was 0.030±0.011 and 0.088±0.031, respectively. Percentage of polymorphic bands and within-breed genetic similarities on the basis of band frequency (BF) and band sharing (BS) for pooled over primers revealed higher genetic similarity in Rathi than Tharparkar cattle population. High estimates of between breed genetic similarities for pooled over primers indicated that either Rathi is having decent from Tharparkar or both the cattle breeds are having common descent. Low value of Index of genetic distances between these two cattle breeds may be due to the fact that Rathi and Tharparkar cattle breeds are the native of Thar Desert in Northwest India. The results of between breed genetic distances also confirm the existence of high degree of genetic similarity between these two breeds of cattle. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 9 : 1204-1209)

Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.