The Project aim.The main goal of this project is to study the regional distribution of dirofilariasis (causative agent: D.immitis, D.repens, and other concurrent filarial species) among canine population in Georgia &Armenia and estimate the disease burden in these countries.
Current status. The problem of dirofilariasis has not been studied sufficiently in Georgia &Armenia. Until recently no proper attention has been drawn to prevention, diagnosis, and treatment of Dirofilaria spp. in animal reservoir in South Caucasus. Dirofilariasis was considered an occasional problem for the region and few identified human cases were thought to be imported from other subtropical and tropical countries. The only survey of canines in different regions of Georgia was conducted by Dr. Tsitskishvili in cooperation with veterinarian clinics in during the period 2005-2008.The main goal of the survey was to reveal high risk zones for the disease and determine ecological and other characteristics of the zones. Almost all clinics from 10 regions and 30 cities/towns of the country participated in the study and provided the requested information on the study subjects. 3887 dogs were examined during the study period and 70 cases identified with confirmed dirofilariasis diagnosis. The study contributed to the understanding of the disease distribution in the country and now dirofilariasis is a well-recognized endemic disease in this geographical area. There was only a single confirmed case of human dirofilariasis in Georgia in 2015 (S. Virsaladze Institute of Parasitology and Tropical Diseases), underestimating the risk of acquiring this parasitic zoonoses for people living in the country. In Armenia dirofilariasis among dogs was evaluated by a survey conducted in 2000. This survey was conducted in regions of Armenia bordering Turkey and Iran and examined a sample of guard dogs at border control/custom points. The survey showed that an estimated 3% of the guard dogs were infected with Dirofilaria spp. Since 2000, not much has been done to study the disease status in the canine population of the country. The disease distribution in the human population of the country also should be evaluated as no reliable information is available on potential disease burden of the disease in the country. Currently there is no ongoing project to study dirofilariasis epidemiology in South Caucasus region.
The project’s influence on progress in this area. 1) Disease burden of dirofilariasis in Georgia &Armenia; 2) Effectiveness of the select study methodology for evaluating dirofilariasis epidemiology in developing countries; 3) Risk factors that are primary contributors for the disease dissemination in Georgia &Armenia; 4) Dirofilariasis infection risk pathways; 5) Dissemination routes in the Caucasus region.
Scope of activities.1) Procedural protocols will be developed and veterinarians trained in performing clinical assessments and collecting baseline information; collecting and handling laboratory samples and data management; 2) Quality of data and samples will be monitored and evaluated throughout the study period; 3) Geographical coordinates of all veterinary clinics and the animal address will be used for GIS mapping; 4) Samples will be examined in parasitological, serological and molecular biology laboratories and an electronic database will be developed for storage and management of laboratory results and associated data; 5) Statistical analysis will be performed using SAS 9.2; Geo-mapping of the results will be performed using GIS 9.3; Risk factors of dirofilariasis will be determined and disease burden in Georgia/Armenia will be assessed; 6) Epidemiological report on dirofilariasis distribution in Georgia/Armenia will be developed; 7) Progress and final reports of the project will be developed; 8) Research manuscripts will be prepared for publication.
Technical approach and methodology. Study population and design. The project will be carried out in all veterinary clinics that are currently operating in Georgia and Armenia. The number of animals from each clinic included in the study will be determined by its representative status at regional level. A cross-sectional study involving one-stage design will be used in the sampling plan. A sampling frame (with no bias in ordering) containing a list of animals to be examined each day, with individual animal identification codes attached to it, will be obtained. Selection of the animal populations to be involved in the study will be based on stratified random sampling (strata will be defined based on animal age, sex, brief preliminary health assessment, and geographic origin).
On-site examination, lab sampling and data collection procedures of the 10% study population.
For each individual animal examined by veterinarian, the following information relevant to epidemiological investigation will be recorded on a data sheet: 1) Individual animal identity; 2) Age; 3) Sex; 4) breed; 5) lifestyle; 6) husbandry.
Methods of Laboratory Research. Blood will be collected from the cephalic vein (5 ml) and stored in tubes with anticoagulant (e.g., EDT; 2.5 ml) or in serum-separating tube (2.5 ml), and later processed for molecular analyses and for parasitological and serological analysis.
Parasitological and Serological Testing: The modified Knott’s technique (KN) and the acid phosphatase histochemical staining test (AP) will be used for microscopic detection and identification of microfilariae in blood smears. The commercial kit WITNESS®Dirofilaria (WT) will be employed for detection of D. immitis circulating antigen in serum.
Molecular Testing: The aim of molecular studies is to identify Dirofilaria species circulating in Georgia & Armenia, to perform sequence analysis of detected species. A variety of PCR-based methodologies are currently used (1-4) to diagnose zoonotic filariae in dogs and combination of PCR and restriction fragment length polymorphism (RFLP) analysis (5, 6) are employed to differentiate wide spectrum of the filarial species. In this project, we will use species-specific semi-nested PCR assay, which is based on amplification of internal transcribed spacer regions ITS1/ITS2 and enables the simultaneous detection and differentiation of filarial species in clinical specimens (7). Particularly, in a first round PCR the entire ITS region will be amplified; in a second round PCR, amplification of ITS1 and ITS2 regions will give the ability to identify D.immitis, D.repens, as well as other filariids -A. reconditum and A.dracunculoides depending on the size of amplification products. The PCR products will be purified and sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc.) in an automated sequencer (ABI-PRISM 377; Applied Biosystems Inc.). All sequences generated will be compared to sequences available in GenBank using Basic Local Alignment Search Tool (BLASTn). Based on the sequencing results, a phylogenetic investigation between species detected in Georgia and Armenia and those reported worldwide will be also carried out. 1. Watts KJ et al. 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. Mol Cell Probes. 1999; 13:425–30; 2. Mar PH, et al. Specific polymerase chain reaction for differential diagnosis of Dirofilaria immitis and Dipetalone mareconditum using primers derived from internal transcribed spacer region 2 (ITS2). Vet Parasitol. 2002; 106:243–52; 3. Rishniw M,et al. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol. 2006; 135:303–14; 4. Latrofa MS, et al. A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs. Acta Trop. 2012; 122:150–4; 5. Nuchprayoon S.,et al. Differentiation of Brugia malayi and Brugia pahangi by PCR-RFLP of ITS1 and ITS2.Southeast Asian J Trop Med Public Health.2003; 34 Suppl 2:67–73; 6. Nuchprayoon S., et al. Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis. Am J Trop Med Hyg.2005; 73:895–900; 7. Catia Ferreira, et al Molecular characterization of Dirofilaria spp. circulating in Portugal. Parasites & Vectors (2017) 10:250
