DIVERSITY AND MORPHOMETRIC DATA OF BLOOD FILARIAL lARVAE IN CARNIVOROUS FROM BULGARIA Текст научной статьи по специальности «Биологические науки»

Фауна, морфология, систематика паразитов

УДК 619:616.995.132

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 im-mitis, D. repens and Acanthocheilonema reconditum. The lengths of microfilariae of the dogs which we related to D. immitis ranged between 237 and 350 ^m and mean value was 297 ^m. Microfilariae’ length related to D. repens ranged from 305 to 420 ^m (mean 365 ^m) and those of А. reconditum were from 223 to 287 (mean 261) ^m. In golden jackals length of microfilariae was from 253 to 344 (mean 309) ^m for D. immitis and 224-285 (mean 250) ^m for А. reconditum. In red foxes microfilariae’ length ranged as follows: from 246 to 320 (mean 281) ^m for D. immitis, from 291 to 355 (mean 321) ^m for D. repens and from 223 to 263 (mean 245) ^m 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

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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 pm and mean value was 297 pm. Microfilariae’ length relat-

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ed to D. repens ranged from 305 to 420 цш (mean 365 цш) and those of А. recon-ditum were from 223 to 287 (mean 261) цт.

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

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Table 1. Metric data of blood filarial larvae in domestic dogs from Bulgaria (in pm) __________________________________ (* larvae from uterus)_______________________________________________

Number of the animal positive for microfilariae Region of Bulgaria Extreme length Mean length Standard deviation length Extreme width Mean width Standard deviation width Species (according to morphometric data)

1 Plovdiv 258,00-308,00 291,652 10,41244 4,80-7,00 6,25 0,47729 D. immitis*

2 Veliko Tamovo 329.63-373.28 345.635 15.5691 7.21-8.24 7.66 0.47560 D. repens

3 (( 237.8-287.32 269.329 16.41350 4.00-6.40 5.32 0.879706 A. reconditum

4 (( 345.75-414.26 388.93 18.65745 7.07-9.09 8.12 0.558861 D. repens

5 (( 334.26-387.57 360.455 19.21834 6.40-8.60 7.497 0.753861 D. repens

6 (( 304.74-390.79 362.003 20.70853 6.08-9.48 7.602 0.863549 D. repens

7 (( 349,83-419,91 381,173 19,24188 6,34-9,00 7,607 0,848539 D. repens

8 (( 272.14-317.33 297.077 13.10419 5.38-8.06 6.944 0.788837 D. immitis

9 (( 336,49-384,23 365,600 14,22298 6,52-8,90 7,718 0,77502 D. repens

10 (( 333,80-372,50 353,114 10,38815 6,40-8,10 7,4538 0,58255 D. repens

11 (( 230,40-279,50 254,033 13,12709 3,00-5,00 3,8917 0,76451 A. reconditum

12 (( 234,71-279,85 256,306 14,29627 4,01-5,65 4,7344 0,62292 A. reconditum

13 (( 243,71-286,47 264,626 15,10909 4,47-5,00 4,8137 0,25890 A. reconditum

14 Plovdiv 278,38-349,85 299,683 23,57763 6,32-8,24 7,3400 0,52082 D. immitis

15 Smolyan 293,12-319,94 302,625 10,38193 6,32-8,06 6,9833 0,72423 D. immitis

16 Sofia 237,00-331,00 294,153 22,99944 6,32-7,81 6,9515 0,49112 D. immitis

17 Sofia 223,00-283,00 259,500 15,90026 5,00-5,83 5,4782 0,38665 A. reconditum

18 Smolyan 278,72-312,39 297,694 10,68609 6,32-8,06 7,2608 0,51617 D. immitis

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Table 2. Metric data of blood filarial larvae in golden jackals from Bulgaria (in pm)

(* larvae from uterus)

Number of the animal positive for microfilariae Region of Bulgaria Extreme length Mean length Standard deviation length Extreme width Mean width Standard deviation width Species (according to morphometric data)

1 Plovdiv 253,00-314,00 290,846 17,18452 6,71-8,60 7,6450 0,63444 D. immitis

2 Plovdiv 289,00-336,00 313,650 14,21739 6,00-8,90 7,5381 0,83395 D. immitis*

3 Burgas 262,86-320,87 303,786 16,93679 6,52-8,61 7,7509 0,84362 D. immitis

4 Plovdiv 244.80-266.80 256.973 5.74984 5.00-7.00 6.1583 0.64309 A. reconditum

5 Plovdiv 224,21-226,30 225,255 1,47785 5,10-6,30 5,7000 0,84853 A. reconditum

6 Burgas 270,41-316,65 297,019 14,60635 5,83-7,81 6,6275 0,53832 D. immitis

7 Plovdiv 233,62-285,23 268,737 14,79816 5,83-7,61 6,9619 0,54494 A. reconditum

8 Burgas 299,80-338,71 324,078 12,96966 6,70-8,60 7,7180 0,71545 D. immitis*

9 Burgas 304.67-344.20 321.637 12.23605 6.02-8.60 7.5062 0.84982 D. immitis

10 Burgas 276.74-334.85 314.073 15.37837 6.08-8.60 7.5822 0.79474 D. immitis

11 Silistra 261.31-325.08 305.043 18.23980 6.00-8.06 7.1037 0.91734 D. immitis

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Table 3. Morphometric data of blood filarial larvae in red foxes from Bulgaria (in pm) ___________________________(* larvae from uterus)_______________________

Number of the animal positive for microfilariae Region of Bulgaria Extreme length Mean length Standard deviation length Extreme width Mean width Standard deviation width Species (according to morphometric data)

1 Plovdiv 264,00-320,00 292,346 14,49122 5,70-8,20 6,8500 0,68250 D. immitis*

2 Sliven 223,00-259,00 242,250 16,68083 5,00-5,38 5,1425 0,16460 A. reconditum

3 Silistra 225,00-263,00 248,454 11,17465 4,12-5,83 5,2027 0,46834 A. reconditum

4 Burgas 290.56-354.72 321.030 20.59336 6.39-8.60 7.4700 0.77210 D. repens

5 Burgas 252,52-293,86 272,591 13,06800 4,45-8,06 6,4436 1,15276 D. immitis

6 Plovdiv 246,44-288,52 268,360 12,68180 5,83-8,06 6,9393 0,71247 D. immitis

7 Plovdiv 261,74-311,49 292,550 13,63330 5,83-8,00 6,8550 0,69457 D. immitis

8 Sliven 258,07-303,94 280,867 12,38214 6,01-8,48 7,3950 0,86365 D. immitis

9 Pleven 194,92-239,09 221,347 14,40255 4,12-6,70 5,4529 0,86969 A. reconditum A. dracuncu-loides

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In golden jackals length of microfilariae was from 253 to 344 (mean 309) pm for D. immitis and 224-285 (mean 250) pm for А. reconditum.

In red foxes microfilariae’ length ranged as follows: from 246 to 320 (mean 281) pm for D. immitis, from 291 to 355 (mean 321) pm for D. repens and from 223 to 263 (mean 245) pm 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. dra-cunculoides. 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.

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.

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.

References

1. Brito A.C., Vila-NovaM.C., Martins Rocha D.A. et al. Prevalence of canine filariasis by Dirofilaria immitis and Dipetalonema reconditum in Maceio, Alagoas State, Brazil. // Cad. Saude Publica. - 2001. - V. 17, № 6. - P. 1497-504.

2. Ciocan R., Darabus Gh., Igna V. Morphometric Study of Microfilariae of Dirofilaria spp. on Dogs // Vet. Med. - 2010. - V. 67, №. 2. - P. 45-49.

3. Demiaszkiewicz A.W., Polanczyk G., Osinska B. et al. Morphometric characteristics of Dirofilaria repens Railliet et Henry, 1911 parasite of dogs in Poland // Wiad. Parazytol. - 2011. - V. 57, №. 4. - P. 253-256.

4. Ettinger S.J., Feldman E.C. Textbook of Veterinary Internal Medicine (7th ed.). W.B. Saunders Company. - 2010.

5. Genchi C. Epidemiology and distribution of Dirofilaria and dirofilariosis in Europe: state of the art. In: Proc. of the Helminthol. Col. - Austria, 2003. - P. 6-11.

6. Genchi C. et al. Guideline for the laboratory diagnosis of canine and feline Dirofilaria infections // Mappe Parassitologiche. - 2005. - № 11. - P. 138-144.

7. Knott J. A method for making microfilarial survey on day blood // Med. Hyg. - 1939. - № 33. - P. 191-196.

8. Magnis J. et al. Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables D. immitis and D. repens species and Acanthocheilonema genus-specific diagnosis // Parasitol. Vectors. - 2013. - V. 6, № 48.

9. Pampiglione S., Canestri Trotti G., Rivasi F. Human dirofilariasis due to Dirofilaria (Nochtiella) repens: a review of world literature // Parassitologia. -1995. - V. 37, №. 2-3. - P. 149-193.

10. Panayotova-PenchevaM., AlexandrovM. Morphometric characteristics of first stage Elaphostrongylus cervi (Nematoda: Protostrongylidae) larvae from Bulgaria // Eur. J. Wildl. Res. - 2008. - V. 54. - P. 771-774.

11. Pratt S.E., Corwin R.M., Selby L.A., Rhoades J.D. Prevalence of Dirofilaria immitis and Dipetalonema reconditum infections in Missouri dogs // J. Am.' Vet. Med. Assoc. - 1981. - V. 179, № 6. - P. 592-593.

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12. Sonin M.D. Osnovy nematodologii. T. 24. Filariati jivotnih i cheloveka i vizivaemie imi zabolevania. - M.: Nauka. 396 s.

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able 4. Reference data of blood filarial larvae in carnivorous (in pm)

Authors Dirofilaria repens Dirofilaria immitis Acantocheilonema reconditum Acantocheilonema dracunculoides

length width length width length width length width

Sonin, 1975 300-360 6.0-8.0 220-290 5.0-7.0 - - 195-230 5.0-5.5

Pratt et al., 1981 — — 255-343 (314 mean) 5,0-7,5 (6,5 mean) 238-295 (257 mean) 3,2-6,5 (4,9 mean) — —

Brito et al., 2001 - - 298,1 7,3 249,2 4,4 - -

Genchi et al., 2005 300-360 6,0-8,0 290-330 5,0-7,0 260-283 4,0 190-247 4,0-6,5

Ciocan et al., 2010 244,66- 360.00 4,5-8,0 — — — — — —

Demiaszkiewicz et al., 2011 296-362 5,0-8,0 - - - - - -

American Heartworm Society, 2012 (http ://www .heartwormsociety. org/v terinary-re source s/canine -guidelines.html) - - 295-325 - 250-288 - - -

Magnis et al., 2013 369,44±10,76 8,87±0,58 301,77±6,29 6,30±0,26 264,83±5,47 4,63±0,52 259,43±6,69 5,09±0,47

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Разнообразие и морфометрические показатели микрофилярий в крови

плотоядных в Болгарии

M. Панайотова-Пенчева, A. Трифонова, Р. Мирчев,

С. Мовсесян***

В 2012-2013 гг. исследовали кровь от домашних собак (Canis familiaris), шакалов (С. aureus) и рыжих лисиц (Vulpes vulpes) из различных областей Болгарии с целью оценки морфометрических показателей обнаруженных личинок филярий. Были проанализированы следующие особенности: форма тела микрофилярий, их длина и максимальная ширина. Согласно морфологическим особенностям выделены три типа личинок. Личинки первого типа имели заостренный передний конец и длинный, тонкий, прямой и острый хвост; второго типа - тупой передний конец и острый хвост, часто заканчивающийся крючком; третьего типа - тупой передний конец и тонкий, острый хвост, в большинстве случаев, изогнутый крючком. Установленные микро-филярии дифференцированы как личинки Dirofilaria immitis, D. repens и Acanthocheilonema reconditum. Длина микрофилярий D. immitis у собак колебалась в пределах 237-350 мкм (в среднем, 297), D. repens - 305-420 (в среднем, 365), А. reconditum - 223-287 мкм (в среднем, 261). У шакалов длина микрофилярий составила D. immitis 253-344 мкм (в среднем, 309) и А. reconditum 224-285 мкм (в среднем, 250). У рыжих лисиц микрофилярии имели длину: D. immitis 246-320 мкм (в среднем, 281), D. repens 291-355 (в среднем, 321) и А. reconditum 223-263 мкм (в среднем, 245).

Ключевые слова: морфометрические особенности, микрофилярии, Dirofilaria immitis, D. repens, Acanthocheilonema reconditum, Canis familiaris, C. aureus, Vulpes vulpes.

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