Guido Favia

Molecular evidence of incipient speciation within Anopheles gambiae s.s. in West Africa

We karyotyped and identified by polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis Anopheles gambiae s.s. samples collected in several African countries. The data show the existence of two non‐panmictic molecular forms, named S and M, whose distribution extended from forest to savannahs. Mosquitoes of the S and M forms are homosequential standard for chromosome‐2 inversions in forest areas. In dry savannahs, S is characterized mainly by inversion polymorphisms typical of Savanna and Bamako chromosomal forms, while M shows chromosome‐2 arrangements typical of Mopti and/or Savanna and/or Bissau, depending on its geographical origin. Chromosome‐2 inversions therefore seem to be involved in ecotypic adaptation rather than in mate‐recognition systems. Strong support for the reproductive isolation of S and M in Ivory Coast comes from the observation that the kdr allele is found at high frequencies in S specimens and not at all in chromosomal identical M specimens. However, the kdr allele does not segregate with molecular forms in Benin.

Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation

Three chromosomal forms of Anopheles gambiae s.s., designated as Bamako, Mopti and Savanna, were studied for diagnostic PCR assays based on the analysis of the X‐linked ribosomal DNA (rDNA). The study was performed on a 1.3 kb fragment containing part of the 28S coding region and part of the intergenic spacer region. The amplified material was cut with fourteen restriction enzymes to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzymes Tru9I and HhaI produced patterns of DNA bands which differentiated Mopti from Savanna and Bamako; moreover, a distinct ‘hybrid’ pattern was recognized in the F1 female progeny from the cross of Mopti with either one of the other two chromosomal forms. The diagnostic significance of the PCR‐RFLP assay was verified on 203 karyotyped females from field samples collected in two villages in Mali and one village in Burkina Faso. Agreement was observed between the chromosomal and the molecular identifications. No ‘hybrid’ molecular patterns were detected even among carriers of rare heterokaryotypes hypothetically produced by crosses between Mopti and Savanna. The results confirm previous observations indicating barriers to gene flow within An. gambiae s.s. and supporting the specific status of the taxonomic units proposed on cytogenetic ground.

Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5′‐most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.

Polymerase chain reaction-identification of Dirofilaria repens and Dirofilaria immitis

On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric.

Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in Mosquitoes (Diptera: Culicidae): Large Polymerase Chain Reaction Survey and New Identifications

Abstract Bacteria of the genus Wolbachia constitute a group of intracellular and maternally inherited micro-organisms that are widespread in arthropods, inducing several reproductive disorders such as cytoplasmic incompatibility in their hosts. Considering relevant biological implications related to the presence of Wolbachia in several insect orders, for example its potential role as mechanism for rapid speciation and as vehicle to drive genetic markers in wild populations of vectors of medical and veterinary interest, we carried out an extensive polymerase chain reaction survey to detect Wolbachia in several species of mosquito belonging to genera involved in the transmission of pathogens. Five species out of 26 tested have shown to be infected; for four of them this is the first evidence of the Wolbachia infection. A phylogenetic analysis was also performed, positioning the five Wolbachia strains in the phyletic subdivision B.

Analysis of the Anopheles gambiae genome using RAPD markers

RAPD analysis technique is used as a rapid and reliable tool for genome analysis in the malaria vector Anopheles gambiae. Using more than eighty different commercially available primers we identified more than sixty different DNA segments that were differentially amplified in different strains of An. gambiae s.s. and An. arabiensis. An estimate of the cytogenetic position of these markers is provided by their hybridization to divisional dot‐blot filters. Potentially useful RAPD markers can be cytogenetically mapped with more precision by in situ hybridization and, as they segregate as dominant markers in a Mendelian fashion, they can also be genetically mapped relative to other genes or rearrangements. Finally, we identified markers for their potential use in the identification of different mosquito strains.

Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectin

Therapy of canine dirofilariois due to Dirofilaria repens is indicated for dogs suffering from clinical signs of this disease, such as dermal swelling, sub-cutaneous nodules and pruritus. It is also important in order to decrease the risk of infection to other dogs and humans in the vicinity of the infected animal when suitable mosquito vectors are present. Combined therapy with the arsenic adulticide melarsomine and the avermectin microfilaricidal doramectin was effective in clearing infection with D. repens in a dog. The number of microfilariae dropped from 17 microl(-1) blood pre-treatment to 7 microl(-1) following the first adulticide injection and reached 0 a day after the microfilaricidal administration. The dog remained negative for D. repens microfilaremia during a follow-up period of 90 days. Euthanasia and necropsy performed 3 months after the initiation of therapy due to a progressive neoplastic disease revealed no evidence of filariae.

Canine dirofilariosis in two cities of southeastern Spain

Several cases of human subcutaneous/ocular dirofilariosis caused by Dirofilaria repens from an area in the southeastern Spain where previous epidemiological studies have shown a very low prevalence of this species in dogs, have been studied in our laboratory. Since the prevalence of this species in dogs did not correspond to the incidence of human cases in the zone studied, a preliminary epidemiological survey was carried out on 114 dog blood samples from two kennels and one veterinary clinic. Knott, polymerase chain reaction (PCR) with specific primers for Dirofilaria immitis and D. repens, and ELISA with adult E/S D. immitis and adult somatic D. repens antigens for the detection of specific IgG, were used. Fifty-three out of the 114 samples analyzed were positive for Knott and/or PCR to D. immitis or to D. repens. D. repens was the species with the highest prevalence, 84.6 and 37.1%, respectively, in each kennel. IgG antibodies against D. immitis and D. repens, were detected in 11 samples which gave negative results to both Knott and PCR. These results demonstrate a very high prevalence of D. repens that could be associated with the increasing incidence of human subcutaneous/ocular cases recently detected in this zone.

Serological assays on eight cases of human dirofilariasis identified by morphology and DNA diagnostics

Specimens of Dirofilaria sp. removed from eight Italian patients were identified as D. repens by morphology and confirmed as such by a PCR-based method of DNA analysis. Blood samples were also drawn from the patients so that two serological tests (ELISA and western blot), one based on the recognition of molecular markers recently identified in the somatic antigenic complex of D. repens, could be evaluated. The antigenic complex used in the ELISA only gave a weak sensitivity. However, the western-blot assays, based on the polypeptide molecular markers, were found to have greater sensitivity and should be useful in detecting human cases of dirofilariasis.

Detection of four Borrelia burgdorferi genospecies and first report of human granulocytic ehrlichiosis agent in Ixodes ricinus ticks collected in central Italy

The presence of Borrelia burgdorferi s.l. and of Ehrlichia phagocytophila group was sought by PCR in Ixodes ricinus collected in a protected area of central Italy. Nymphs (n = 1475, gathered in 295 pools of 5 nymphs each) and adult ticks (n = 28) were examined. B. burgdorferi s.l. was detected in 13.8% of the nymph pools; of these, 63.4% were infected by B. valaisiana, 26.8% by B. afzelii, 7.3% by B. garinii, and 2.5% by B. burgdorferi s.s. Only a single adult male tick proved to host B. afzelii. The agent of human granulocytic ehrlichiosis (HGE) was detected in 2.7% of the nymph pools. Two HGE agent-positive nymph pools were also found to be positive for B. garinii and for B. afzelii, respectively. This is the first report from central Italy of the finding of the HGE agent in ticks.