Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host

The mosquito-borne filarial worm, Dirofilaria immitis, causes heartworm disease in dogs. Detection of this parasite in its mosquito intermediate host currently involves dissection and microscopic examination for larval stages. Although this method is used commonly as a screening tool for epidemiological surveys, it lacks both sensitivity and specificity. In this study, a more efficient PCR- and probe-based diagnostic assay was developed. The target selected for this assay is a segment of the 16 S rRNA gene. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, equivalent to DNA derived from one third stage larva (L(3)), but does not detect 100 ng (10 000-fold excess) of the purified DNA from several other filarial nematodes, including Dirofilaria striata, Dirofilaria tenuis, Dipetalonema reconditum, Wuchereria bancroftii, Brugia pahangi, B. malayi, Onchocerca volvulus or Loa loa. This assay also detects one L(3)of D. immitis, the minimal biological unit of infection, in a pool of 200 mosquito heads. This assay can serve as a highly specific and sensitive tool for efficiently screening the large numbers of mosquitoes to determine, with statistical validity the seasonal transmission pattern of D. immitis in a locality prior to designing a rational preventive medication program for that parasite.     

A simple method to design PCR primer to detect genomic DNA of parasites and its application toDirofilaria immitis

A simple method to design a polymerase chain reaction (PCR) primer for parasites of interest was developed usingDirofilaria immitisas a test sample. The method involved the cloning and sequencing of randomly amplified DNA of the parasite, and designing a primer based on the resulting DNA sequence. Using the primer, DNA fragments of the expected length were amplified by a regular PCR with genomic DNA ofDirofilaria immitis.     

The development and evaluation of a single step multiplex PCR method for simultaneous detection of Brugia malayi and Wuchereria bancrofti

A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites, Brugia malayi and Wuchereria bancrofti, from blood samples and mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188bp (BM) and 129bp (WB) DNA fragments, specific to B. malayi and W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both B. malayi and W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Synthesis, cytotoxicity, and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diamines

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis. This species is often associated with drug resistance, and the conventional treatments exhibit high toxicity for patients. Therefore, the search for new antileishmanial compounds is urgently needed since there is no vaccine available. In this study, using the in vitro traditional drug screening test, we have analyzed the effects of a series of diaminoalkanes monoprotected with t-butyloxycarbonyl (BOC) against L. amazonensis. Among the 18 tested compounds, 6 exhibited antileishmanial activity (2, 7–9, 17, and 18). Best IC₅₀ values (10.39 ± 0.27 and 3.8 ± 0.42 μg/mL) were observed for compounds 17 and 18 (H₂N(CH₂)nNHBoc, n = 10 and 12), respectively. Although those compounds had higher lipophilicity as indicated by their cLog P values, compound 17 was very toxic. Determination of the selective indexes indicated that 50% of the active compounds were very toxic for HepG2 cells. However, compounds 2, 8, and 18 had good lipophilicity and were less toxic among all polyamine derivatives tested. The chemical properties of antileishmanial diamine derivatives, such as lipophilicity and cytotoxicity, are relevant factors for the design of new drugs. A higher lipophilicity is likely to improve the chances of reaching this intracellular parasite.     

Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group

This study used group-specific PCR combined with SNaPshot minisequencing for species identification within the Lactobacillus casei group. The L. casei group-specific PCR primer pair was designed using the rpoA gene sequence. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 63 strains of L. casei group species. The results showed that the group-specific PCR could assign Lactobacillus strains into the L. casei group, and the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for species identification of members of the L. casei group.     

Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti

Plasmid libraries enriched for microsatellites were generated in the tick, Ixodes scapularis and in the mosquito Aedes aegypti. Libraries were enriched for genomic DNA containing (AC)_n, (AG)_n, (ATG)_n, (CAG)_n, (TAG)_n, (AAT)_n, (CTGY)_n or (GATA)_n motifs. Clones containing each motif were sequenced in both species for PCR primer design. In I. scapularis, most primers amplified a single locus and alleles varied in the number of microsatellite repeats and segregated as codominant markers. In contrast (AC)_n, (TAG)_n and (GATA)_n microsatellite loci extracted from Ae. aegypti appeared to be members of multigene families. A primer pair designed to amplify a particular TAG locus instead amplified many independently segregating loci, some of which did not contain TAG microsatellites. Alleles at the TAG loci segregated as dominant markers and there was limited evidence for length variation among alleles. These results suggest that microsatellite loci are not universally abundant in arthropod genomes nor do alleles always segregate as codominant markers.     

Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus

Melanization is an effective defence reaction used by mosquito hosts to kill malarial and filarial worm parasites. Although phenoloxidase (PO) has long been considered to be the key enzyme in the biosynthesis of melanotic material in insects, there is no direct evidence verifying its role in parasite melanization. To elucidate the role of PO in the melanization of microfilariae (mf) by mosquitoes, a double subgenomic Sindbis (dsSIN) recombinant virus was used to transduce Armigeres subalbatus mosquitoes with a 600 base antisense RNA targeted to the highly conserved copper-binding region of an Ar. subalbatus PO gene. Compared with controls, haemolymph PO activity in mosquitoes transduced with antisense RNA was significantly reduced. When these mosquitoes were challenged with Dirofilaria immitis mf, the melanization of mf was almost completely inhibited. These data verify that PO is an essential component of the biochemical pathway required for the melanization of parasites, and that the dsSIN expression system represents a useful tool in the functional analysis of endogenous gene expression in mosquitoes.     

5 S ribosomal spacer sequences of some filarial parasites: comparative analysis and diagnostic applications

We have amplified by PCR the sequences of the 5 S ribosomal spacer of Setaria labiatopapillosa and Foleyella furcata. After sequencing, these sequences have been compared with those of Dirofilaria immitis and Dirofilaria repens. Two major goals have been achieved: (i) the establishment of a multiplex PCR-based diagnostic assay, applicable to identify the four species in vertebrate and invertebrate hosts; (ii) the identification, in S. labiatopapillosa and F. furcata, of a canonical spliced leader 1 (SL1) sequence, so confirming that only D. repens, of the filarial parasites so far studied, shows a peculiar SL1 sequence. The PCR assay here developed and the analysis of the 5 S ribosomal spacer, can further improve both epidemiological and molecular analysis of these filarial species.     

Discovery of Potent Small-Molecule Inhibitors of Multidrug-Resistant Plasmodium falciparum Using a Novel Miniaturized High-Throughput Luciferase-Based Assay

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P. falciparum parasites that stably express cytoplasmic firefly luciferase. After initial optimization, we tested two different types of compound libraries: known bioactive collections (Library of Pharmacologically Active Compounds [LOPAC] and the library from the National Institute of Neurological Disorders and Stroke [NINDS]) and a library of uncharacterized compounds (ChemBridge). A total of 12,320 compounds were screened at 5.5 μM. Selecting only compounds that reduced parasite growth by 85% resulted in 33 hits from the combined bioactive collection and 130 hits from the ChemBridge library. Fifteen novel drug-like compounds from the bioactive collection were found to be active against P. falciparum. Twelve new chemical scaffolds were found from the ChemBridge hits, the most potent of which was a series based on the 1,4-naphthoquinone scaffold, which is structurally similar to the FDA-approved antimalarial atovaquone. However, in contrast to atovaquone, which acts to inhibit the bc₁ complex and block the electron transport chain in parasite mitochondria, we have determined that our new 1,4-napthoquinones act in a novel, non-bc₁-dependent mechanism and remain potent against atovaquone- and chloroquine-resistant parasites. Ultimately, this study may provide new probes to understand the molecular details of the malaria life cycle and to identify new antimalarials.The number of episodes of malaria worldwide is estimated to be around half a billion per year (30). Malaria caused by the parasite Plasmodium falciparum results in approximately 1 to 3 million deaths per year, mostly among young children under the age of 5. The recent and continuing emergence of parasite strains resistant to antimalarials is a major challenge and has been directly linked to the increase in the global mortality associated with this disease (29). As such, the discovery and development of novel antimalarial therapeutics is essential.The development and pursuit of chemical high-throughput screening (HTS) methods can provide new bioactive molecules against the malaria parasite and also may help identify new “druggable” targets for future therapeutic development. HTS can utilize two basic approaches: (i) biochemical target-based screens or (ii) whole-organism-based screens. Biochemical screens must have a predefined target, a protein that typically is necessary for parasite survival. These targets normally must be recombinantly expressed (or purified from native sources) and utilized in a robust activity or binding assay to screen for inhibitors. One drawback in biochemical screening is that small-molecule hits may not be able to reach their protein target in a live cell assay because of poor cell permeability or the presence of drug efflux pumps. Whole-organism screening is advantageous because all small-molecule hits act directly against a live cell, in this case a parasite, and all possible targets expressed in that cell are included in a single screen. However, frequently the target of each hit is unknown and requires identification, which may involve complex purification protocols and/or genetic validation.Currently, there are several methods used to screen for parasite viability in culture. To date, the main methods for detecting parasite growth are based on a measurement of DNA content. The most widely used growth assay is the [³H]hypoxanthine incorporation assay (9). This assay relies on the incorporation of tritiated hypoxanthine into DNA during the development of daughter parasites. Several groups have adapted the use of fluorescent DNA stains such as SYBR green or 4′,6′-diamidino-2-phenylindole (DAPI) to measure parasite growth (1, 7, 19, 26, 28). A shortcoming of these fluorescence-based DNA detection screens is the modest signal-to-background ratio of 3- to 9-fold, which may result in a high number of false positives and false negatives. In addition, flow-cytometric assays have been developed that rely on measuring the DNA content of parasitized red blood cells (RBCs) using a fluorescent DNA stain such as propidium iodide or YOYO (23, 35). One shortcoming currently is that these flow cytometry-based assays are difficult to scale up for the HTS of many thousands of compounds.Given this historical reliance on DNA-based screens to quantify parasite content, we set out to develop a new screen with optimal properties for high-throughput screening. We used a transgenic strain of parasites stably expressing cytoplasmic firefly luciferase (3D7-luc) (18) to measure the growth of intracellular parasites during their erythrocytic phase using luminescence as the readout. This luciferase assay is similar to one previously described (8). However, we now have adapted this luciferase-based assay to a 384-well plate HTS, providing a novel tool for P. falciparum screening that displays a high dynamic signal-to-noise range compared to that of traditional assays. We tested two compound collections, the Library of Pharmacologically Active Compounds (LOPAC) and the library from the National Institute of Neurological Disorders and Stroke (NINDS), that have known targets and/or confirmed bioactivity. We also used a collection of uncharacterized compounds from the ChemBridge DiverSet library to facilitate the discovery of novel antimalarial scaffolds. The ChemBridge library contains small sets of analogs based on common pharmacophores to aid in the determination of structure-activity relationships (SAR).In this study, we report the screening of 12,320 compounds against the erythrocyte life cycle of the P. falciparum parasite using our luciferase-based assay. A number of compounds containing novel drug scaffolds were identified with potent activity against both standard drug-sensitive 3D7 (although somewhat resistant to sulfadoxine [34]) and a multidrug-resistant parasite line, TM90C2B.     

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

Several cDNA clones with similarity to α-amylases have been characterized from a library made from adult female salivary gland RNA isolated from the vector mosquito, Aedes aegypti. The corresponding gene, designated Amylase I (Amy I), is expressed specifically in the proximal-lateral lobes of the adult female salivary gland, a pattern overlapping that of another gene, Mal I, involved in carbohydrate metabolism. The deduced amino acid sequence of Amy I indicates that this gene encodes a protein, approximate M_r= 81,500, that appears to be a novel member of the amylase gene family. The mosquito protein contains a putative signal peptide for secretion and several consensus sites for asparagine-linked glycosylation. The Amy I protein shows significant similarity to invertebrate and vertebrate amylases including the conservation of four reactive and substrate binding sites. However, the amino-terminal region of the Amy-I protein is unique to the mosquito. Similarity with the Drosophila melanogaster protein is evident only after the first 260 amino acids in the mosquito sequence. The identification of this gene and its expression pattern adds to the observed relationship between spatial-specific gene expression in the female salivary glands and the specific feeding mode of the adult mosquito.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the γ-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Differentiation of seventeen genospecies ofAcinetobacterby multiplex polymerase chain reaction and restriction fragment length polymorphism analysis

The 17 described genomic species (DNA groups) of the genusAcinetobacter, including the type strains of the seven named species, were studied by using a multiplex PCR. The multiplex PCR assay combined two primer sets (rA1 and rA2 forrecAgene target; rib1 and rib2 for 16S rDNA sequence) in a single reaction. Restriction analysis with two enzymes (MboI andHinfI) of the enzymatically amplified products allowed identification of all genospecies. This technique proved to be a rapid and reliable method for the identification of theAcinetobactergenomic species, including the closely related DNA groups (1, 2, 3, 13). The results of this study suggest that the proposed method can be used for the identification ofAcinetobacterspp. and as such may help to elucidate the ecology and clinical significance of the different species of this genus.     

Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H, K-ATPase

We purified a compound with strong inhibitory effect on H⁺, K⁺-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-β- -glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC₅₀ of the compound for H⁺, K⁺-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K⁺. Pentagalloylglucose had relatively weak inhibitory effects for Mg⁺-ATPase (IC₅₀: >10 μmol/l) and Na⁺, K⁺-ATPase (IC₅₀: 2.7 μmol/l). Pentagalloylglucose also inhibited the accumulation of [¹⁴C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 μmol/l histamine (IC₅₀: 7.8 μmol/l) and 1 mmol/l dbc-AMP (IC₅₀: 10 μmol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H⁺, K⁺-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions

Anisakis simplex, a nematode from the family Anisakidae, is a parasite of fish and mammals. It is a casual agent of a human disease called anisakiosis. We found that the assay based on PCR amplification of the ITS-1–5·8 S–ITS-2 fragment of rDNA and subsequent restriction fragment length polymorphism, previously described on the basis of A. simplex isolated solely from one geographical region, can be used as a general test for identification of this worm species. The restriction patterns analysed for four restriction enzymes were found to be identical in the case of allA. simplex individuals isolated from as different geographical regions as Baltic Sea, Norwegian Sea, Bering Sea and Sea of Okchotsk. Moreover, our results support the previously proposed hypothesis, based on the studies of isoenzymes, that there is a remarkable genetic homogeneity within A. simplex from different geographical regions.     

Recovery of Coccidioides immitis from blood and abscess fluid using the BacT/Alert system

We report the recovery of Coccidioides immitis from the blood and abscess fluid of two separate patients by using two automated blood culture systems. In the first case, an aspirate from a neck abscess containing C. immitis spherules was serially diluted and inoculated into liquid media used by the BacT/Alert and the Bactec NR660 blood culture systems. BacT/Alert bottles inoculated with 10⁵, 10⁴, 10³, 10², 10, and two spherules produced a positive signal at 19, 24, 35, 42, 57, and 62 h postinoculation, respectively. Bactec NR660 bottles containing >10² spherules and 10 spherules produced a positive signal after approximately 72 and 96 h of incubation, respectively. In the second case, a blood specimen incubated in BacT/Alert blood culture broth was signaled positive after 82 h of incubation. No organisms were detected by Gram stain of the broth, but C. immitis grew after blind subculture. Our observations demonstrate that these rapid blood culture systems are capable of supporting growth of C. immitis. To our knowledge, this report is the first to detect C. immitis by these blood culture systems.     

Transgenic,inducible RNAi in megakaryocytes and platelets in mice

Summary Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)‐regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet‐regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet‐on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV‐rtTA mice) display inducible reporter gene expression with unusual and near‐complete efficiency in megakaryocytes and platelets. To test whether the CMV‐rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet‐regulated short hairpin RNA (shRNA) targeting Bcl‐x_L, a pro‐survival Bcl‐2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl‐x_L in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl‐x_L levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl‐x_L, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.     

Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5′ nuclease PCR assays

This study describes fluorogenic 5′ nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi andE.intestinalis . The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10⁴ and 10⁻¹ spores per PCR sample. The coefficients of variation were ≤5·2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10⁴ spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.     

Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles)

The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fiftyseven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested.