Towards a rapid molecular identification of the common phlebotomine sand flies in the Mediterranean region

The present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors.     

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

Digital Agenesia in Martina Franca Donkey Foal: A Case Report

A 4-day-old male Martina Franca donkey foal was evaluated for a forelimb alteration. Clinical examination and radiographs revealed the agenesia of the distal digit. Biochemical parameters were normal, and ultrasonographic evaluation did not identify any relievable organ alteration. Karyotype study revealed an abnormality on chromosome 1. The foal was discharged with a distal limb bandage in which a palmar splint was applied. A poor prognosis for the functionality of the limb was given. In endangered species, such as the Martina Franca donkey, the excessive inbreeding could result in an increase in genetic disorders. These findings shed new light on the possible pathogenesis of the digital dysgenesia. The study of the karyotype could be a useful approach to detect genetic alterations that could or could not be expressed in the animal, especially in endangered species in which a risk of an excessive inbreeding is considerable. These defects should be considered in the choice and selection of the breeders.     

In vitro antifungal susceptibility of Malassezia pachydermatis from dogs with and without skin lesions

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections.     

Effects of milk feeding, frequency and concentration on weaning and buffalo (Bubalus bubalis) calf growth, health and behaviour

Growth, weight at birth and daily weight gain (DWG) on 12 water buffalo calves, starting from 6 days of age until completion of weaning, was investigated in this study. Different feeding regimens were given to two groups of animals with regard to daily milk replacer: (1) group 1 (G1) received a double concentration in single administration; whereas (2) group 2 (G2) received the same amount of milk replacer split twice daily. Blood samples were collected from each calf on days 6, 30, 60 and 90 to evaluate acute phase proteins (haptoglobin), bactericide activity, lysozime, total protein content and biochemical parameters. No differences were observed between the two groups in terms of dry matter intake, feed efficiency and live body weight at the end of the study. Interestingly, a significantly (P?<?0.05) reduced DWG was observed earlier in G1 (day 45) than in G2 (day 60). Gastrointestinal disorders were not recorded throughout the experimental period, and no significant differences were recorded between the two groups for all considered parameters. This study confirms the possibility of utilising one daily administration of milk replacer in water buffalo calf during weaning. This new approach facilitates calves management, without interfering with calves growing performances.     

Cross-sectional study on acrylamide hemoglobin adducts in subpopulations from the European Prospective Investigation into Cancer and Nutrition (EPIC) Study

Acrylamide exposure was investigated in subgroups of the EPIC study population (510 subjects from 9 European countries, randomly selected and stratified by age, gender, and smoking status) using hemoglobin adducts of acrylamide (HbAA) and its primary metabolite glycidamide (HbGA). Blood samples were analyzed for HbAA and HbGA by HPLC/MS/MS. Statistical models for HbAA and HbGA were developed including body mass index (BMI), educational level, and physical activity. A large variability in acrylamide exposure and metabolism between individuals and country groups was observed with HbAA and HbGA values ranging between 15-623 and 8-377 pmol/g of Hb, respectively. Both adducts differed significantly by country, sex, and smoking status. HbGA values were significantly lower in high alcohol consumers than in moderate consumers. With increasing BMI, HbGA in nonsmokers and HbAA in smokers decreased significantly. In the assessment of potential health effects related to acrylamide exposure, country of origin, BMI, alcohol consumption, sex, and smoking status should be considered.     

Cutaneous sterile pyogranuloma/granuloma syndrome in a dog

A case of canine cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) with generalized asymmetrical alopecia and plaques is described. Results from histopathologic examination were suggestive of SPGS and failed to demonstrate any etiological agent. A systematic approach on how to definitively rule out infectious skin diseases is discussed.     

Application of 10% imidacloprid/50% permethrin to prevent Ehrlichia canis exposure in dogs under natural conditions

Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is the most known canine tick-borne disease (TBD) spread throughout the world. Preventing tick bites is a priority to reduce the risk of TBDs and it was the aim of the present study to evaluate the efficacy of a combination of imidacloprid 10% and permethrin 50% (ImPer) (Advantix; Bayer AG, Germany) in a spot-on formulation to control CME under field conditions. On January-March 2005, 845 dogs from two kennels in southern Italy (kennels of Bari (KB)- and Ginosa (KG)), with a history of tick infestation were initially tested by serology and PCR assay for E. canis infection. Data on Leishmania infantum infection were also available from a previous study carried out on the same dog population. One hundred twenty-six dogs (14.9%) presented anti-E. canis antibodies with a relative prevalence of 15.6% (n=65 dogs in KB) and 14.2% (n=61 dogs in KG). Five hundred thirty-five animals found negative both for E. canis and L. infantum infections were enrolled in three groups (Group A--treated with ImPer once a month; Group B--treated every 2 weeks; and Group C--untreated control animals) and monitored for E. canis infection by serology and PCR in November 2005 (first follow-up) and in March 2006 (second follow-up). The E. canis infection was serologically revealed, at the first and/or second follow-up, in 26 animals from Group C in KB and KG (mean incidence density rate (IDR), 13.24%) while in none of the animals from Group A (KB and KG) and only in one animal from Group B (IDR 1.13%) in KG. The final protection efficacy of ImPer ranged from 95.57% to 100% in Groups B and A. At PCR only 15 dogs from KG were positive for Rickettsiales only at the first follow-up and at the sequence analysis two (both in Group C) revealed 100% homology with E. canis sequences while 13 with Anaplasma platys. Four out of 13 A. platys PCR-positive dogs were also seropositive for E. canis at one or both follow-ups. ImPer, by virtue of its repellent and acaricidal activity against ticks, has been shown to be efficacious to prevent E. canis infection in treated dogs living under natural conditions in endemic areas.     

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs,humans and wildlife in south‐east Spain

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south‐east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%–100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.