STUDIES ON SOME BLOOD PARASITES OF DOGS IN ASSIUT GOVERNORATE WITH SPECIAL REFERENCE TO MICROFILARIAE

Animal Health Research Institute

Assiut Regional Laboratory

Studies on some blood parasites of dogs

in Assiut Governorate with special reference to microfilariae

(With 5 Tables and 2 Plates)

By

G.A. Taher, GEHAN M. Sayed and M.I. Arafa

(Received at 15/3/2010)

دراسات على بعض طفيليات الدم في الکلاب بمحافظة أسيوط وخاصه

يرقات الفيلاريا

جمال امين طاهر , جيهان محمد سيد ، محسن إبراهيم عرفه

تم عمل مسح طفيلي على عدد ١٤٥ کلب (١٢٥ من الکلاب الضالة من مناطق مختلفة في محافظة أسيوط، و٢۰ من الکلاب المستوردة الشبرد التابعة لقسم الشرطة بمدينة أسيوط) وذلک لاستبيان معدل الإصابة بطفيليات الدم فى هذه الحيوانات خلال الفترة من مارس2009م حتى فبراير2010م. بلغت نسبة الإصابة الکلية بنوع واحد أو أکثر من طفيليات الدم ٥۱‚٦٥٪موزعة کالتالي: يرقات الفيلاريا ٤۸‚١٤٪ ، بابيزيا کانز٤٤‚٢٣٪وهيباتوزون کانز ٥٥‚٢٧٪. کان معدل العدوى فى الکلاب الضالة بطفيليات الدم المختلفة ٧۲ ٪ في حين بلغ فى الکلاب الأجنبية ۲٥ ٪ (دلاله احصائية عالية). وقد اوضحت الدراسة زيادة معدل الاصابة بطفيليات الدم المختلفة مع تقدم عمر تلک الحيوانات. وتم تصنيف الميکروفيلاريا إلى نوعين هما ديروفيلاريا إميتس وديروفيلاريا ريبنس وقد بلغت نسبة الاصابة بهما فى الکلاب الضالة ٢‚٣%و13,6%على التوالى بينما لم يتم العثور على اى من النوعين في الکلاب الأجنبية. وقد استخدمت أربعة طرق مختلفة لتشخيص والتعرف على أنواع الميکروفيلاريا المختلفة وهي: مسحات الدم الرطبة (الغير جافة) مسحات الدم السميکة، طريقة الترسيب الشعري وطريقة اختبار نوت واتضح أن الفحص الطفيلي بطريقة نوت هو أدق الطرق للتشخيص والتعرف على أنواع الميکروفيلاريا المختلفة حيث بلغت نسبة دقتها في تشخيص الميکروفيلاريا٦٦‚٦٦٪. کانت أعلى نسبة إصابة بطفيليات الدم المختلفة خلال فصلى الصيف ٥‚۹٥٪ والربيع ٥٧‚ ٧۸ ٪ کانت نسبة الإصابة أقل في فصل الشتاء ۲٥٪ وقد وجدت دلاله احصائية عالية لنسبة الاصابة الموسمية وقد يفسر ذلک بالارتباط الوثيق بين هذه الطفيليات وانتشار وتکاثر الحشرات الناقلة للطفيليات في هذا الوقت من السنة.

Summary

A parasitological survey was carried out on 145 dogs (125 stray dogs from different localities in Assiut Governorate and 20 foreign breed Shephered dogs in the security unit at Assiut city) to estimate the prevalence of different blood parasites during the period from March 2009 till February 2010. Out of 145 examined dogs 95 (65.51%) were infected with one or more parasitic species, 21 (14.48%) were positive for microfilariae, 34 (23.44%) with Babesia canis and 40 (27.58%) with Hepatozoon canis. The infection rate of stray dogs with different blood parasites was 72% while in foreign breed dogs was 25% (statistically highly significant). Mixed infection by more than one blood parasite was detected in 19 dogs. Two typesof microfilariae were detected in examined animals: Dirofilaria immitis and Dirofilaria repens and their infection rate in stray dogs was 3.2% and 13.6% respectively. No microfilariae were detected in the blood films of foreign breed ones. Four methods were used in this study for the diagnosis of microfilariae: direct wet method, thick blood smears, capillary sedimentation technique and modified Knott's method. The modified Knott's test was the most reliable test, its sensitivity was 66.66%. The present work cleared that the incidence of infection of dogs with different blood parasites increased with age in both breeds. The highest rate of infection with different blood parasites was recorded during Summer (95.5%) and Spring (78.6%) while the lowest rate in Winter (25%).Seasonal variations were found statistically highly significant and this may be due to correlation between the infestation rate and the flourishing of the insect population and tick infestation during this period of the year.

Key words: Dog, blood parasites, microfilariae, prevalence.

Introduction

Dogs not only act as a dangerousreservoir host for most of the parasitic diseases but also they play a role in transmission of a number of important pathogens among domestic animals and human being that deteriorate their health and lead to enormous losses.

Dogs suffer from a variety of blood parasites as: Babesia (intra-erythrocytic protozoa), Hepatozoon (intra - leukocytic protozoa), Trypanosoma (inter-erythrocytic protozoa) and filarial worms(Gadahi  et al., 2008).

Heartworm disease in dogs and related canines is caused by a filarial nematode (a large thread-like round worm), Dirofilaria immitis. The adult worms live in the right side of the heart (right ventricle) and adjacent blood vessels (pulmonary arteries), and because of their location, are commonly called dog heartworms. It is particularly common in the tropics and subtropics. It seems that all breeds of dogs are equally susceptible (Retnasapathy and San 1976). It is also of public health importance because it often produces nodules in the pulmonary parenchyma and subcutaneous tissue or even the eyes of humans (Seo Youngwoo et al., 2001).

Dirofilaria repens is commonly found in the subcutaneous tissue of dogs in Southern Europe, Africa and Asia (Zahler et al., 1997). Subcutaneous and subconjunctival migration of immature nematodes has been reported in human beings (Pampiglione et al., 1995).

Both dirofilariasis due to D. immitis and D. repens are transmitted by the bite of anopheline and culicine mosquitoes harboring the infective third stage larvae.

In epizootiologic surveys of dirofilariasis in dogs, various methods for determining infection status have been used. These have included microscopic examination of blood smears, blood sample concentration techniques (e.g Knott's test) for detectingcirculating microfilariae, examination for identifying adult worms at necropsy, radiographic and angiographic evaluations (Knight, 1977 and Thrall      et al., 1980).

Babesia spp. parasitize erythrocytes, causing anemia in dogs. Different species exist with varying host specificity. B. canis and          B. gibsoni are two organisms commonly known to infect dogs. B. canis is commonly known to infect dogs and it is comparatively highly pathogenic for both young and old dogs. It is found throughout Asia, Africa, Europe, the Middle East, and North America (Levine, 1985).

Hepatozoon is a genus of protozoa belonging to phylum Apicomplexa. Dogs are known to be susceptible to infection by Hepatozooon canis and Hepatozoon americanum. The detection of capsule-like gamonts in the cytoplasm of neutrophils in blood smears is helpful for diagnosing hepatozoonosis (Baneth et al., 2001).

Both Babesia and Hepatozoon parasites are transmitted by ticks in which sexual stages occur (Levine, 1961).

In Egypt dog's blood parasites were studied previously by several authors (Botros et al., 1973, Sakla, 1975, Amer, 1986, Mahmoud, 1998 and Abd -el Rahim, 1998).

The aim of the present work is to throw further light on some of the blood parasitic diseases infecting dogs in Assiut Governorate with special reference to microfilariae which have pubic health hazards (zoonotic importance). In addition, to evaluate the sensitivity of different techniques used for diagnosis of microfilariae.

Materials and methods

Animals

            During the period from March 2009 to February 2010, one hundred and fourty five dogs (125 stray dogs from different localities in Assiut Governorate and 20 foreign breed Shephered dogs in the security unit at Assiut Governorate, upper Egypt) aged between 6 months and over 7 years old were used in this study.

Sampling:

            5ml of blood was drained from each dog from the cephalic vein in vacum tubes for blood parasite examination.

Parasitological examination: Four parasitological techniques were done for detection of the most reliable one for diagnosis of microfilariae.

- Fresh blood was examined for detection of motile microfilariae with direct wet smear method (Levine, 1985).  

- Haematocrit centrifugation technique and Knott's technique were done for detection of mild microfilaraemia (Lowernce & Thomas, 1987).

- Blood films (thin and thick) were prepared from fresh blood, fixed by absolute methyl alcohol, stained by Giemsa stain and examined by light microscope for detection of different blood parasites.

- The detected parasites were microphotographed and identified according to the description given by Levine (1985) and Soulsby (1986).

- The average dimensions of various anatomical regions were determined by eyepiece micrometer. Seasonal investigation of the detected blood parasites was done.

Postmortem examination: Infected dogs were thoroughly examined for adult Dirofilaria immitis and Dirofilaria repens during necropsy in the Veterinary Medical Hospital of Assiut Governorate.

Statistical analysis: Obtained data were subjected to a software program (SPSS) according to Borenstein et al. (1997).

Results

Out of 145 examined dogs 95 (65.51%) were infected with one or more parasitic species, 21 (14.48%) were positive for microfilariae, 34 (23.44%) with Babesia canis and 40 (27.58%) with Hepatozoon canis. The infection rate of stray dogs with different blood parasites was 72% while in foreign breed dogs was 25%. Mixed infection by more than one blood parasite was detected in 19 dogs(18 stray dogs and 1 foreign breed dog).

Two typesof microfilariae were detected in examined animals: Dirofilaria immitis and Dirofilaria repens and their infection rate were 2.75% and 11.72% respectively. Both typesof microfilariae were detected in the blood films of stray dogs only (Table 1). Mixed infection with the two types of microfilariae was recorded in 3 stray dogs.

Data concerning the seasonal variation and the effect of age on the rate of infection with different blood parasites are shown in (Table 2 and 3).

Blood parasites detected in the peripheral blood of dogs are:

1- Dirofilaria immitis microfilariae:

Out of 125 examined stray dogs, 4 (3.2%) were positive for D. immitismicrofilariae. The microfilaria was unsheathed. It is characterized by a tapered cranial end, straight caudal end and a straight body shape. The body cavity was filled with a large number of minute oval deeply stained nuclei appeared as one mass interrupted at certain anatomical landmarks (Plate 1, Table 4). Their total length ranged from 291.6 - 346.66 µm (325 µm) and their width ranged from 5.4 - 7.2 µm         (6.75 µm).

2- Dirofilaria repens microfilariae:

It was detected in 17 (13.6%) of examined stray dogs. The microfilaria was non sheashed and lie in graceful curves in stained blood smears. It had a blunt cranial end, a button shaped caudal end and a cresentic shaped body. A single row of somatic nuclei is faint stained throughout the whole length of the body of the microfilariae which appeared as spiral shaped line. The somatic nuclei clump together but not mask the anatomical landmarks in most cases of the microfilariae. Their total length ranged from 289 - 330 µm (310 µm) and their width ranged from 3.75: 6.25 µm (5.75 µm).

The microfilarial landmarks were detected in the form of interruptions in the column of nuclei (Plate 1, Table 4).

The result of the comparative sensitivity of different techniques used for the detection of the microfilariae indicated that modified Knott’s technique is the most sensitive technique (66.66%) followed by wet blood film technique (42.85%), thick blood film (33.33%) and haematocrit centrifugation (28.57%) (Table 5).

Routine P.M examinations of 100 dogs at the veterinary clinical hospital in AssiutUniversity failed to detect any adult worms.

3- Babesia canis:

Out of 145 examined dogs, 34 (23.44%) were positive for Babesia canis. The incidence was 24% and 20% among stray and foreign breeds dogs respectively (Table 1)

Various forms of Babesia canis have been detected in the peripheral blood of dogs. Ovoid shaped mostly single or multiple. It had one marginally located chromatin mass in addition to central vacuole. It measured 3.5: 4.7μm in length by 2.1: 2.8 μm in width (average 3.8: 2.4 μm) (Plate 2, Fig. 2).Amoeboid forms measured 3.5: 4.8 μm in length by 2: 3.4 μm in width. Centrally located vacuoles are commonly seen (Plate 2 Fig. 1). Pyriform or pear shaped forms are commonly detected. They are found in pairs and less frequently in single infection. They measured from 3.5 μm in length by 2: 2.6 μm breadth (Plate 2 Fig. 3).

4- Hepatozoon canis

Out of 145 examined dogs 40 (27.58%) dogs were infected with Hepatozoon canis.The incidence was 31.2% and 5% among stray dogs and foreign breed ones respectively (Table 1).

Morphological observations of gametocytes:

A- Macrogametocytes

These are ovoidal bodies with blunt rounded ends measuring 9: 11 μm long and 5: 7.5 μm wide. The centrally located nucleus is large compact strippled and spherical. The cytoplasm is granular and stains dark blue with Giemsa stain. Macrogametocytes are surrounded by delicate fine capsule. (Plate 2 Fig. 4,5).

B- Microgametocytes

They are elongate, rectangular, sausage shaped bodies with blunt rounded ends measuring 8.5-11.2 μm long by 3.4- 4.8 μm wide, they stain pale blue with Giemsa stain. The nucleus is large, compact and strippled and is usually located at one pole of the parasite. The microgametocytes are enveloped by delicate fine capsules. On several occasions microgametocytes were found to lie within the nuclear substance of the polymorphnuclear leucocytes. (Plate 2, Fig. 6). Sometimes the parasite was found to lie within the cytoplasm of the host cell. Free gametocytes devoid of their capsules were usually encountered anywhere out side the host cell.

Table 1: Prevalence of different blood parasites in examined dogs.

                  Data were found statistically highly significant.

                                                   Chi square 16.85                    P(probability) <0.0001

Table 2: Relation between the age and the prevalence rate of infection with dog's blood parasites

           Table 3: Seasonal prevalence of dog's blood parasites.

            Highly significant statistical variations between seasons and prevalence of infection.

            Chi square 50.133                                                                  P (probability) <0.00001