Effectiveness of the spot-on combination of moxidectin and imidacloprid (Advocate®) in the treatment of ocular thelaziosis by Thelazia callipaeda in naturally infected cats

Background: The present study evaluated the therapeutic effectiveness of moxidectin 1.0% (w/v) and imidacloprid 10% (w/v) (Advocate® spot-on solution for cats, Bayer Animal Health) against natural infections with the eyeworm Thelazia callipaeda in cats. This study was conducted as a GCP, negative-controlled, blinded and randomised field study in privately owned cats living in an area in southern Italy where T. callipaeda is enzootic. Methods: The study was carried out in 30 cats (19 females and 11 males, aged from 8 months to 5 years, weighing 1.2-5.2 kg) of different breeds, naturally infected by T. callipaeda. At study inclusion (Day 0), animals were physically examined and the infection level was assessed by examination of both eyes for clinical score and live adult T. callipaeda count. Each cat was weighed and randomly assigned to one of the treatment groups (G1: Advocate, G2: untreated control). Clinical assessments and T. callipaeda adult counts were performed on Day 14. At the study completion visit on Day 28, clinical assessments and counts of T. callipaeda adults and larvae were performed. All cats were daily observed by their owners and general health conditions were recorded during the entire period of the study. Results: The primary effectiveness variable was the percentage of animals in G1 group (Advocate) showing a complete elimination (parasitological cure) of adult eye worms at Day 14 and Day 28. The effectiveness of the treatment in the G1 group was 93.3 and 100% at Day 14 and Day 28, respectively, when compared to group G2. Total worm count reduction from both eyes for Advocate was 96.3% on Day 14 and 100% on Day 28. Clinical data were confirmed by the examination of conjunctival pouch flushing. An overall reduction in the number of cats with lacrimation and conjunctivitis was observed following treatment despite the fact that in a few cats treated with Advocate clinical signs persisted due to the chronic nature of the disease. Conclusions: Based on the results of the present trial, a single dose of Advocate was found to be safe and highly effective in the treatment of natural T. callipaeda infection in cats

The sticky resting box, a new tool for studying resting behaviour of Afrotropical malaria vectors

Background: Monitoring densities of adult mosquito populations is a major challenge in efforts to evaluate the epidemiology of mosquito-borne diseases, and their response to vector control interventions. In the case of malaria, collection of outdoor-resting Anophelines is rarely incorporated into surveillance and control, partially due to the lack of standardized collection tools. Such an approach, however, is increasingly important to investigate possible changes in mosquito behaviour in response to the scale up of Insecticide Treated Nets and Indoor Residual Spraying. In this study we evaluated the Sticky Resting Box (SRB) - i.e. a sticky variant of previously investigated mosquito Resting Box, which allows passive collection of mosquitoes entering the box – and compared its performance against traditional methods for indoor and outdoor resting mosquito sampling.<p></p> Methods: Daily collections were carried out in two neighbouring villages of Burkina Faso during rainy season 2011 and dry season 2012 by SRB located indoors and outdoors, and by Back-Pack aspiration inside houses (BP) and in ad hoc built outdoor pit-shelters (PIT).<p></p> Results: Overall, almost 20,000 Culicidae specimens belonging to 16 species were collected and morphologically identified. Malaria vectors included Anopheles coluzzii (53%), An. arabiensis (12%), An. gambiae s.s. (2.0%) and An. funestus (4.5%). The diversity of species collected in the two villages was similar for SRB and PIT collections outdoors, and significantly higher for SRB than for BP indoors. The population dynamics of An. gambiae s.l. mosquitoes, as obtained by SRB-collections was significantly correlated with those obtained by the traditional methods. The predicted mean estimates of An. gambiae s.l. specimens/sampling-unit/night-of-collections was 6- and 5-times lower for SRB than for BP indoors and PIT outdoors, respectively.<p></p> Conclusions: Overall, the daily performance of SRB in terms of number of malaria vectors/trap was lower than that of traditionally used approaches for in- and outdoor collections. However, unlike these methods, SRB could be set up to collect mosquitoes passively over at least a week. This makes SRB a promising tool for passively monitoring anopheline resting populations, with data presented here providing guidance for how to set up SRB-based collections to acquire information comparable to those obtained with other methods.<p></p&gt

In silico karyotyping of chromosomally polymorphic malaria mosquitoes in the Anopheles gambiae complex

Chromosomal inversion polymorphisms play an important role in adaptation to environmental heterogeneities. For mosquito species in the Anopheles gambiae complex that are significant vectors of human malaria, paracentric inversion polymorphisms are abundant and are associated with ecologically and epidemiologically important phenotypes. Improved understanding of these traits relies on determining mosquito karyotype, which currently depends upon laborious cytogenetic methods whose application is limited both by the requirement for specialized expertise and for properly preserved adult females at specific gonotrophic stages. To overcome this limitation, we developed sets of tag single nucleotide polymorphisms (SNPs) inside inversions whose biallelic genotype is strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural variation. Beginning with principal components analysis (PCA) of population samples, applied to windows of the genome containing individual chromosomal rearrangements, we classified samples into three inversion genotypes, distinguishing homozygous inverted and homozygous uninverted groups by inclusion of the small subset of specimens in Ag1000G that are associated with cytogenetic metadata. We then assessed the correlation between candidate tag SNP genotypes and PCA-based inversion genotypes in our training sets, selecting those candidates with >80% agreement. Our initial tests both in held-back validation samples from Ag1000G and in data independent of Ag1000G suggest that when used for in silico inversion genotyping of sequenced mosquitoes, these tags perform better than traditional cytogenetics, even for specimens where only a small subset of the tag SNPs can be successfully ascertained

Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex

Background: Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods: We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results: For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions: The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.[Figure not available: see fulltext.

A new species in the major malaria vector complex sheds light on reticulated species evolution

Complexes of closely related species provide key insights into the rapid and independent evolution of adaptive traits. Here, we described and studied Anopheles fontenillei sp.n., a new species in the Anopheles gambiae complex that we recently discovered in the forested areas of Gabon, Central Africa. Our analysis placed the new taxon in the phylogenetic tree of the An. gambiae complex, revealing important introgression events with other members of the complex. Particularly, we detected recent introgression, with Anopheles gambiae and Anopheles coluzzii, of genes directly involved in vectorial capacity. Moreover, genome analysis of the new species allowed us to clarify the evolutionary history of the 3La inversion. Overall, An. fontenillei sp.n. analysis improved our understanding of the relationship between species within the An. gambiae complex, and provided insight into the evolution of vectorial capacity traits that are relevant for the successful control of malaria in Africa

Leishmania tarentolae and leishmania infantum in humans, dogs and cats in the pelagie archipelago, southern italy

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection

Inversion genotyping in the Anopheles gambiae complex using high-throughput array and sequencing platforms

Chromosomal inversion polymorphisms have special importance in the Anopheles gambiae complex of malaria vector mosquitoes, due to their role in local adaptation and range expansion. The study of inversions in natural populations is reliant on polytene chromosome analysis by expert cytogeneticists, a process that is limited by the rarity of trained specialists, low throughput, and restrictive sampling requirements. To overcome this barrier, we ascertained tag single nucleotide polymorphisms (SNPs) that are highly correlated with inversion status (inverted or standard orientation). We compared the performance of the tag SNPs using two alternative high throughput molecular genotyping approaches vs. traditional cytogenetic karyotyping of the same 960 individual An. gambiae and An. coluzzii mosquitoes sampled from Burkina Faso, West Africa. We show that both molecular approaches yield comparable results, and that either one performs as well or better than cytogenetics in terms of genotyping accuracy. Given the ability of molecular genotyping approaches to be conducted at scale and at relatively low cost without restriction on mosquito sex or developmental stage, molecular genotyping via tag SNPs has the potential to revitalize research into the role of chromosomal inversions in the behavior and ongoing adaptation of An. gambiae and An. coluzzii to environmental heterogeneities

Hyperendemic Dirofilaria immitis infection in a sheltered dog population: an expanding threat in the Mediterranean region

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott's test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe

Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities

Background: Leishmania tarentolae is a protozoan isolated from geckoes (Tarentola annularis, Tarentola mauritanica), which is considered non-pathogenic and is transmitted by herpetophilic Sergentomyia spp. sand flies. This species occurs in sympatry with Leishmania infantum in areas where canine leishmaniasis is endemic. In the present study, we investigated the circulation of L. tarentolae and L. infantum in sand flies, dogs and lizards in a dog shelter in southern Italy, where canine leishmaniasis by L. infantum is endemic. Methods: Sheltered dogs (n = 100) negative for Leishmania spp. (March 2020) were screened by immunofluorescence antibody test (IFAT) using promastigotes of both species at two time points (June 2020 and March 2021). Whole blood from dogs, tissues of Podarcis siculus lizards (n = 28) and sand flies (n = 2306) were also sampled and tested by a duplex real-time PCR (dqPCR). Host blood meal was assessed in sand flies by PCR. Results: Overall, 16 dogs became positive for L. infantum and/or L. tarentolae by IFAT at one or both sampling periods. One canine blood sample was positive for L. infantum, whilst two for L. tarentolae by dqPCR. At the cytology of lizard blood, Leishmania spp. amastigote-like forms were detected in erythrocytes. Twenty-two tissue samples, mostly lung (21.4%), scored molecularly positive for L. tarentolae, corresponding to 10 lizards (i.e., 35.7%). Of the female Sergentomyia minuta sampled (n = 1252), 158 scored positive for L. tarentolae, four for L. infantum, and one co-infected. Two Phlebotomus perniciosus (out of 29 females) were positive for L. tarentolae. Engorged S. minuta (n = 10) fed on humans, and one P. perniciosus, positive for L. tarentolae, on lagomorphs. Conclusions: Dogs and lacertid lizards (Podarcis siculus) were herein found for the first time infected by L. tarentolae. The detection of both L. tarentolae and L. infantum in S. minuta and P. perniciosus suggests their sympatric circulation, with a potential overlap in vertebrate hosts. The interactions between L. tarentolae and L. infantum should be further investigated in both vectors and vertebrate hosts to understand the potential implications for the diagnosis and control of canine leishmaniasis in endemic areas. Graphical abstract: [Figure not available: see fulltext.