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DIVERSITY AND MORPHOMETRIC DATA OF BLOOD FILARIAL
lARVAE IN CARNIVOROUS FROM BULGARIA
M. Panayotova–Pencheva*, A. Trifonova**, R. Mirchev**,
S. Movsesyan***
* Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev St., Block 25, 1113 Sofia, Bulgaria, e-mail: marianasp@abv.bg
** National Research Station of Game Management, Biology and Pathology, 5 «Iscarsko shoes» Blvd., 1528 Sofia, Bulgaria, e-mail: apth@abv.bg
*** Center of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, 33 Leninskii Prospect, 119071, Moscow, Russian Federation, e-mail: movsesyan@list.ru
During the period 2012–2013 investigations of blood samples from domestic dogs (Canis familiaris), goldon jackals (Canis aureus) and red foxes (Vulpes vulpes) originated from different regions of Bulgaria were performed aiming to document diversity and morphometric characteristics of established filarial larvae. Following features of microfilariae were analyzed: peculiarities in the shape, body length and maximum body width. According to the morphological characteristics three types of larvae were observed. Larvae of the first type were with pointed anterior end and long, thin, straight and sharp tail; those of the second type were with obtuse anterior end and sharp tail, often ending as an umbrella handing; larvae of the third type were with obtuse anterior end and thin, sharp tail, in the most cases curved as a hook. Established filarial larvae were related to following species – Dirofilaria immitis, D. repens and Acanthocheilonema reconditum. The lengths of microfilariae of the dogs which we related to D. immitis ranged between 237 and 350 µm and mean value was 297 µm. Microfilariae’ length related to D. repens ranged from 305 to 420 µm (mean 365 µm) and those of А. reconditum were from 223 to 287 (mean 261) µm. In golden jackals length of microfilariae was from 253 to 344 (mean 309) µm for D. immitis and 224–285 (mean 250) µm for А. reconditum. In red foxes microfilariae’ length ranged as follows: from 246 to 320 (mean 281) µm for D. immitis, from 291 to 355 (mean 321) µm for D. repens and from 223 to 263 (mean 245) µm for А. reconditum.
Keywords: morphometric features, microfilariae, Dirofilaria immitis, D. repens, Acanthocheilonema reconditum, Canis familiaris, C. aureus, Vulpes vulpes.
Dirofilaria immitis, D. repens, Acanthocheilonema (Dipetalonema) dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae [8]. Some of them as D. repens and D. immitis are species of zoonotic importance [9]. D. immitis occurs in the lung arteries, occasionally adults migrate to the right heart [4] and causes so-called heartworm disease in dogs and cats. The canine dirofilariosis produced by it is usually diagnosed on the basis of identifying antigens of adult female. Parasitosis caused by other filariae such as: A. reconditum, D. repens, A. dranculoides, Cercopithifilaria grassi, Brugia malayi, B. pahangi and B. ceylonensis, are characterized by persistent microfilaremia and negative results at the adult Dirofilariae specific antigens detection test [5]. Sometimes there are no possibilities of performing immunological tests as diagnostic way. At that time for instance microfilariae of Dirofilaria sp. must be differentiated morphologically from other species of filariae [2]. Available data on filarial larvae’ size vary considerably in the literature [8]. These variations could be due to different factors such as parasite and host species, their environment etc.
In this connection was made the aim of the present work namely to perform blood analyses of different carnivorous from Bulgaria and documented morphometric characteristics of established filarial larvae.
Materials and methods
During the period 2012–2013 blood samples from stray and domestic dogs were investigated for a presence of microfilarial larvae. The dogs originated from the following parts of Bulgaria: 76 from the region of Sofia, 54 from the region of Veliko Tarnovo, 41 from the region of Plovdiv and 58 from the region of Smolyan. During the same period helminthological necropsies and blood samples’ investigations of 19 carnivorous from different regions of the country were performed, 10 of them were from golden jackals (Canis aureus L.), 8 from red foxes (Vulpes vulpes L.) and 1 from dogs (Canis familiaris L.). The wild animals were hunted according to the Low of the hunting and preserve of the game (2000) and the domestic dog was given dead by his owners. The blood samples from 187 stray and watch dogs were collected by the staff of Foundation «Four paws» who was working on the Project «Care for homeless animals». Microfilariae in the blood samples were detected by the modified Knott method [7].
Between 10 and 25 microfilariae from each Dirofilaria positive animal or such with microfilaraemia were analyzed. The analyzed features were body length, maximum body width and shape (form of the front end and tail).
The larvae were photographed on a light microscope «Amplival» supplied with a specially adapted «Logitech Quick Cam Pro 4000» camera. Simultaneously obtained pictures of an object-micrometer parts (Reichert, division 200 parts on 2 mm) were used to calibrate the software measurements. Morphometric parameters were evaluated by the image analyzing computer program Image-Pro Plus – Version 6 as described in Panayotova-Pencheva and Alexandrov [10]. Obtained data were statistically analyzed by the software Statistica 7 (StatSoft, Inc).
Results and Discussion
According to the morphological characteristics three types of larvae were observed. Larvae of the first type were with pointed anterior end and long, thin, straight and sharp tail (Fig. 1); those of the second type were with obtuse anterior end and sharp tail, often ending as an umbrella handing (Fig. 2); larvae of the third type were with obtuse anterior end and thin, sharp tail, in the most cases curved as a hook (Fig. 3). Metric data of the larvae from the different hosts are shown in the Tables 1, 2 and 3. Taking into consideration larval morphologycal and metric characteristics (Tables 1–3) and comparing them with data by other authors [1–3, 6, 8, 11, 12] which presented in Table 4, we considered that established microfilariae were of three different species – D. immitis, D. repens and Acanthocheilonema reconditum.
In some cases we observed particular structure at the anterior end of the larvae which we related to А. reconditum (Fig. 3), probably this was the hook described by some other authors [6].
The length of microfilariae of the dogs which we related to D. immitis ranged between 237 and 350 µm and mean value was 297 µm. Microfilariae’ length related to D. repens ranged from 305 to 420 µm (mean 365 µm) and those of А. reconditum were from 223 to 287 (mean 261) µm.
Fig. 1. Filarial larva in the blood sample from a golden jackal related to Dirofilaria immitis
Fig. 2. Filarial larva in the blood sample from a domestic dog related to Dirofilaria repens
Fig. 3. Filarial larva in the blood sample from a domestic dog related to Acanthocheilonema reconditum. Arrows: a hook at the anterior end
Table 1. Metric data of blood filarial larvae in domestic dogs from Bulgaria (in μm) (* larvae from uterus)
Table 2. Metric data of blood filarial larvae in golden jackals from Bulgaria (in μm) (* larvae from uterus)
Table 3. Morphometric data of blood filarial larvae in red foxes from Bulgaria
(in μm) (* larvae from uterus)
We hope, the presented morphometric characteristics of these nematodes can be useful in primary diagnostics of filariid infections in carnivorous both in Bulgaria and other countries. In golden jackals length of microfilariae was from 253 to 344 (mean 309) µm for D. immitis and 224–285 (mean 250) µm for А. reconditum.
In red foxes microfilariae’ length ranged as follows: from 246 to 320 (mean 281) µm for D. immitis, from 291 to 355 (mean 321) µm for D. repens and from 223 to 263 (mean 245) µm for А. reconditum.
According to referent values showed in Table 4 metric data concerning filarial larvae from a red fox № 9 could be related to species A. reconditum and A. dracunculoides. According to Magnis et al. [8] due to the overlapping size ranges of A. dracunculoides and A. reconditum biochemical or molecular methods are required to distinguish these two species. Taking into consideration above mention facts we leave this question for decision in next studies.
It could be stated in conclusion that founding on morphometric characteristics of blood microfilariae of the present studies we relate them to the following three species: D. immitis, D. repens and A. reconditum. In domestic dogs in the most cases we have established microfilariae of the species D. repens followed by D. immitis and A. reconditum. In golden jackals they are arranged as follow: D. immitis and A. reconditum, and in red foxes: D. immitis, A. reconditum and D. repens.
This work was supported by the Ministry of Education, Youth and Science (Bulgaria), Grant No DDVU-02/62/2010. It was fulfilled in cooperation of BAS and RAS and connected with a research task of National Research Station of Game Management, Biology and Pathology, Bulgaria.
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Table 4. Reference data of blood filarial larvae in carnivorous (in μm)
© 2015 The Author(s). Published by All-Russian Scientific Research Institute of Fundamental and Applied Parasitology of Animals and Plants named after K.I. Skryabin.
This is an open access article under the Agreement of 02.07.2014 (Russian Science Citation Index (RSCI)) and the Agreemnt of 12/06/2014 (CABI org / Human Sciences section)