Efficacy of 65% permethrin applied to dogs as a spot-on against Phlebotomus perniciosus

Leishmania infantum is a protozoan parasite causing leishmaniosis, a visceral disease transmitted by the bites of sand flies. As the main reservoir of the parasite, dogs are the principal targets of control measures against this disease, which affects both humans and dogs. Several studies have revealed the usefulness of topical insecticide treatment (collars, spot-ons and sprays) in reducing the incidence and prevalence of L. infantum. The present study was designed to test the efficacy of 65% permethrin applied to dogs as a spot-on against the sand fly vector Phlebotomus perniciosus. The duration of the desired effects was also estimated to help design an optimal treatment regimen. Twelve dogs assigned to treatment (n=6) and control (n=6) groups were exposed to sand flies once a week over a seven-week period. Repellent and insecticidal efficacies were estimated and compared amongst the groups. Our findings indicate satisfactory repellent, or anti-feeding, effects lasting 3 weeks and short-term insecticidal effects lasting 2 weeks after initial application. Accordingly, we recommend the use of this product every 2-3 weeks during the active phlebotomine sand fly period to protect dogs against the bites of P. perniciosus.     

The importance of canine leishmaniosis in non-endemic areas, with special emphasis on the situation in Germany

This review article summarizes the situation of canine leishmaniosis in Germany. Published case studies on infections with Leishmania (L.) infantum in either humans or dogs are analyzed. Diagnosed cases of infections by Leishmania spp. in humans and animals are not a notifiable disease in Germany or other European countries. Taking this into consideration one may assume that there might be a significant gap between the analyzed and reported cases and the infectious status within the country. The reported case studies and results from surveys indicate that the majority of all L. infantum infections are acquired during travelling in endemic regions, predominantly the Mediterranean region. However there are cases reported from human infections and growing number of cases in dogs, where the case history may indicate an autochthonous infection within Germany, a country within a non-endemic region. The current data from entomological field studies proved the presence of two phlebotomine sand fly species. Phlebotomus (P.) mascittii, an anthropophilic sand fly species and P. perniciosus a proven vector of L. infantum. The impact from a growing leishmania-positive dog population within Germany, the distribution of at least two sand fly species, one with vector potential in the light of climate change and other non-vectorial transmissions are summarized.     

Evaluation of the efficacy of a topically administered combination of imidacloprid and permethrin against Phlebotomus perniciosus in dog

The phlebotomine sand fly Phlebotomus perniciosus is one of the main vectors of Leishmania infantum, responsible for human and canine leishmaniasis in the Mediterranean Basin. The objective of this study was to evaluate the repellent and insecticidal efficacy of imidacloprid 10% (w/v)/permethrin 50% (w/v) spot-on against sand flies (P. perniciosus) on dogs. The dogs used in this trial were laboratory-bred beagles: eight were impregnated with the solution (treated group), while the other eight were left untreated (control group). On day 0 the animals in the treatment group received 0.1 ml/kg body weight of the combination imidacloprid/permethrin spot-on. Dogs were exposed for 1h to about 100 female sand flies at weekly intervals for a period of 4 weeks, on day 1, 7, 14, 21, and 28 after applying the product. The repellency criterion was based on the feeding rate of sand flies in the treated compared to the untreated group. The insecticidal efficacy criterion was based on comparison of the survival rate of sand flies between the two groups. The product had an insecticidal efficacy on female sand flies of 53.2% (day 1), 49.4% (day 7), 15.1% (day 14), 13.2% (day 22), and 2.9% (day 29). The product showed a repellent effect of 97.7% (day 1), 96.3% (day 7), 96.5% (day 14), 92.7% (day 22), and 74.0% (day 29). Within the first week of application the insecticidal effect was significant; however it did not surpass 50%. On the other hand, the product showed a potent anti-feeding effect of over 90% during the first 3 weeks of this trial. Therefore, the application of this product every 3 weeks would be a good tool to significantly reduce sand fly bites over the period of transmission of vectorial diseases such as leishmaniasis and several arbovirosis such as Toscana virus.     

A survey on canine leishmaniasis and phlebotomine sand flies in central Italy

Zoonotic visceral leishmaniasis (ZVL) is a vector-transmitted zoonosis caused by the parasitic protozoan Leishmania infantum. Bloodsucking sand flies of the subfamily Phlebotominae are the obligatory insect hosts, and the dog is the only domestic reservoir.This study reports data from a survey of canine infection and sand fly phlebotomine monitoring in the province of Perugia in central Italy. The overall seroprevalence in a total of 100 dogs tested was 8% (95% confidence interval: 3.8-15.6%). Data analysis revealed that serological positivity was statistically associated with age (p-value = 0.03) and the area where dogs lived. Standard blacklight traps employed for sampling Culicoides midges in bluetongue disease surveillance were used in phlebotomine monitoring. A total of 5698 sand flies were collected and the two species, Leishmania competent vectors, were identified, Phlebotomus perfiliewi (50%) and Phlebotomus perniciosus (30%).     

Prevention of sand fly attack by topical application of a permethrin/pyriproxyfen combination on dogs

Dogs are the primary domestic reservoir of Leishmania infantum, the parasite responsible for most cases of human visceral leishmaniasis. A strategy for the control of leishmaniasis would be to inhibit the sand fly bite. A study was designed to measure the prevention of the sand fly attack by spraying a combination of permethrin and pyriproxyfen on dogs artificially exposed to the vector of leishmaniasis. Eight dogs were individually challenged with 100 female sand flies for 1 hour on Days -7, 0, 7, 14, 21, and 28. Four dogs were randomly assigned to a control group and four dogs were treated with topically applied permethrin/pyriproxyfen on Day 0. After each exposure, sand flies were collected, counted, and scored as fed or unfed. Efficacy of the combination for prevention of feeding was based on the number of unfed sand flies (dead or alive). The combination of permethrin/pyriproxyfen demonstrated a significant (P <.05) repellent effect against Phlebotomus perniciosus bites as soon as it was sprayed on the dogs, and its repellent efficacy lasted at least for 28 days. The combination product provided significant (P <.05) knockdown activity against challenge with sand flies for 21 days in adult dogs and 14 days in puppies. These findings indicate that adult animals in endemic areas should be sprayed with the permethrin/pyriproxyfen product at 3- to 4-week intervals, and young dogs should be sprayed at approximately 2-week intervals, to prevent sand fly attack.     

Predicted Distribution of Visceral Leishmaniasis Vectors (Diptera: Psychodidae; Phlebotominae) in Iran: A Niche Model Study

Visceral leishmaniasis (VL) is an important vector‐borne disease in Iran. Till now, Leishmania infantum has been detected from five species of sand flies in the country including Phlebotomus kandelakii, Phlebotomus major s.l., Phlebotomus perfiliewi, Phlebotomus alexandri and Phlebotomus tobbi. Also, Phlebotomus keshishiani was found to be infected with Leishmania parasites. This study aimed at predicting the probable niches and distribution of vectors of visceral leishmaniasis in Iran. Data on spatial distribution studies of sand flies were obtained from Iranian database on sand flies. Sample points were included in data from faunistic studies on sand flies conducted during 1995–2013. MaxEnt software was used to predict the appropriate ecological niches for given species, using climatic and topographical data. Distribution maps were prepared and classified in ArcGIS to find main ecological niches of the vectors and hot spots for VL transmission in Iran. Phlebotomus kandelakii, Ph. major s.l. and Ph. alexandri seem to have played a more important role in VL transmission in Iran, so this study focuses on them. Representations of MaxEnt model for probability of distribution of the studied sand flies showed high contribution of climatological and topographical variables to predict the potential distribution of three vector species. Isothermality was found to be an environmental variable with the highest gain when used in isolation for Ph. kandelakii and Ph. major s.l., while for Ph. alexandri, the most effective variable was precipitation of the coldest quarter. The results of this study present the first prediction on distribution of sand fly vectors of VL in Iran. The predicted distributions were matched with the disease‐endemic areas in the country, while it was found that there were some unaffected areas with the potential transmission. More comprehensive studies are recommended on the ecology and vector competence of VL vectors in the country.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Characterization of <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions

In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3’end of 18S and at the 5’start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.     

Validation of the usefulness of 26S rDNA D1/D2, internal transcribed spacer, and intergenic spacer 1 for molecular epidemiological analysis of Macrorhabdus ornithogaster

Macrorhabdus ornithogaster (MO) is an infectious fungus that causes gastric damage in birds. In this study, we established nested and seminested polymerase chain reaction (PCR) methods that specifically amplify the domain D1/D2 region (D1/D2) of 26S ribosomal DNA (rDNA), internal transcribed spacer (ITS) of rDNA, and intergenic spacer (IGS) 1 region from avian feces. Phylogenetic analysis of MO collected from Japanese pet birds showed little genetic variation; analysis based on these regions did not distinguish between host species order, differences in MO shape, or host gastrointestinal symptoms. These regions were found to be unsuitable for molecular epidemiological studies of MO and further investigation into other genetic regions is required.     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

狼尾草属牧草rDNA的ITS序列分析

对来自福建、江苏、海南等15份狼尾草属牧草的5.8SrDNA、ITS1及ITS2片段进行克隆及序列分析,并登录于GenBank数据库,采用DNAMAN、CLUSTALX、MEGA等软件分析其遗传关系聚类图。结果显示,克隆的目的片段长度为573～586bp,聚类结果总体能较好地反应狼尾草属牧草之间的遗传距离,其中杂交狼尾草和细茎杂交狼尾草可能存在着同种异名的现象。     

PCR amplification and sequencing of ITS1 rDNA of Culicoides arakawae

The first internal transcribed spacer (ITS1) of nuclear ribosomal DNA from Culicoides arakawae was amplified by PCR, cloned and sequenced. The wDNAsis software was used to analyze the ITS1 sequences of C. arakawae and other nine species of Culicoides, which were obtained from GenBank and EMBL databases. For all species, the lengths of the ITS1 were 316-469 bp, and the G+C contents were 26.79-34.53%. Based on the lengths of the ITS1 sequences, the 10 Culicoides species could be divided into two groups. The first group consisted of C. arakawae, C. albicans, C. cubitalis, C. pulicaris and C. punctatus, and the second group comprised C. impunctatus, C. nubeculosus, C. variipennis, C. grisescens and C. imicola. The lengths for the first group were 316-347 bp and the second group were 457-469 bp. C. arakawae belonged to the first group by its ITS1 sequence length. Sequence analysis revealed that C. arakawae was genetically more similar to the first group than it was to the second group, consistent with results based on sequence length. The alignment of ITS1 (the alignment length was 500 bp including the gaps) sequences showed that there was a highly conserved region, which was between 288 and 388 bp, except for a few insertions and substitutions. These findings have important implications for the molecular identification of C. arakawae, for studying its molecular genetics and epidemiology, and for studying the molecular systematics of Culicoides.     

桑树ITS序列测定及特点的初步分析

以蒙桑 (M .mongolicaSchneid)为试验材料 ,采用PCR产物直接测序法测定了其核糖体基因转录内间隔区 (InternalTranscribedSpacer ,IST)的全序列 ,分析了桑树该序列的特点 ,指出了该序列在桑树分子系统学及分子进化研究中潜在的重要作用     

分子技术在蜜蜂白垩病病原鉴定和诊断上的应用

蜜蜂白垩病是一种由球囊菌属（Ascosphaera）的真菌寄生而引起蜜蜂幼虫死亡的传染性疾病，对蜂群的群势发展影响极大，近几年又呈现出大面积爆发的趋势。但目前对于该病的诊断和病原鉴定多依赖于典型症状，方法极不可靠。本文介绍了可用于分子鉴定和诊断的的研究基础，探讨利用球囊菌属的各病原rDNA的ITS1—5．8S-ITS2序列资料，分别设计出高特异性引物，用PCR技术扩增出可用于鉴定病原物种的分子标记，从而构建一套快速、准确诊断该病的分子技术。     

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.     

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis.

METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results.

RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR.

CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.     

PCR-based species-specific amplification of ITS of Mecistocirrus digitatus and its application in identification of GI nematode eggs in bovine faeces

Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes.     

Molecular typing of sand fly species (Diptera, Psychodidae, Phlebotominae) from areas endemic for Leishmaniasis in Ecuador by PCR-RFLP of 18S ribosomal RNA gene

Surveillance of the distribution of sand fly species is important for prediction of the risk and expansion of Leishmania infection in endemic and surrounding areas. In the present study, a simple and reliable method of typing New World Lutzomyia species circulating in endemic areas in Ecuador was established by using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. PCR-RFLP of 18S ribosomal RNA (rRNA) genes with the restriction enzyme AfaI and subsequently HinfI successfully identified seven sand fly species in nine endemic areas in Ecuador. Although intraspecific genetic-diversity affecting the RFLP-patterns was detected in a species, the patterns were species specific. The method promises to be a powerful tool for the classification of New World Lutzomyia species.     

Characterization of sand fly breeding sites in district Malakand,Khyber Pakhtunkhwa,Pakistan, and evaluation of risk factors for cutaneous leishmaniasis in the region

Present study was carried to determine the sand fly species composition, breeding sites ecology, seasonal abundance, and spatial distribution in district Malakand, Khyber Pakhtunkhwa, Pakistan. In addition, risk factors associated with cutaneous leishmaniasis (CL) were also evaluated. Survey of indoor and outdoor habitats was carried out using sticky traps in 31 villages of Dargai and Batkhela tehsils of Malakand. Soil from habitats of adult and immature sand flies was analysed. Questionnaire-based household survey was also performed in these villages to assess risk factors associated with CL. Soil samples from selected CL positive households were analysed for its contents. Additionally, clinicoepidemiological data from local health centres was examined for the year 2019. Total of 3,140 sand flies belonging to 18 species were collected. Phlebotomus sergenti was the most abundant species (38.16%). Its abundance had a strong positive correlation with mean monthly relative humidity and negative correlation with average temperature. Phlebotomus sergenti and Phlebotomus papatasi were abundant at an elevation ranging from 320 to 1,120 m above sea level and in agricultural lands near human settlements. Flight height preference apparatus collected maximum sand flies at 30 cm (1ft) above the ground and all species associated negatively with height. Soil analysis from habitats of adult and immature flies showed that highest mean number of adults and immatures were recorded from silt loam which carried highest concentrations of K₂O, Mg, Ca, and Zn. Number of immature sand flies correlated moderately (r = .7, p < .05) with K₂O soil concentrations. There was significant similarity between organic matter contents in soil samples from positive breeding sites and CL households (Wilcoxon rank-sum test, p = .1976). In multivariate analysis model for CL risk factors, age (26–35 and >35 years), knowledge of leishmaniasis, living in a middle and upper class, preachers visit to villages, and assumption that Afghan refugees are more prone to CL were significant. CL patient's archived data from health centres showed that majority of patients had lesions on face and hands. Patient's influx was highest in February and March.     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.