Serological study of Dirofilaria immitis in urban dogs of Urmia using modified knot and rapid antigen tests

Introduction

D. immitis is the most important zoonotic species of the genus Dirofilaria in the world (Navarrete et al., 1968; Schlotthauer et al., 1969; Toomes et al., 1983). Dirofilariasis can be an infectious zoonotic disease when humans are bitten by culicid mosquitoes harboring infective third-stage larvae (microfilariae), due to D. immitis. This nematode is a common filarial worm of dogs and cats in the world, accounts for the most number of human infection with over 400 cases recorded (Pampiglione et al., 1995; Vakalis et al., 1997; Brown et al., 1993). Dirofilariasis has been reported as an emerging zoonosis in many countries world-wide such as Iran (Azari-Hamidian, 2007).

D. immitis or heartworm is more important than other filaria in dogs because it can cause some clinical signs (Brown et al., 1993). This worm lives in right ventricle, pulmonary artery and posterior vena cava and its microfilaria is found in the peripheral circulation (Eslami, 1998).

Infection to D. immitis in dogs was reported from different regions of the world (Hatsushika et al., 1992). The first infectious was recorded by Travassol in 1921 (Ettinger, 2010). Dirofilariasis is common in dogs in Iran with infection rates 16/1% (Ranjbar et al., 2011).

D. immitis was first reported from a dog in Iran in 1969 (Sadighian, 1969). Then infection to heartworm was reported from different areas of Iran including: Ardabil (Bokai et al., 1998), Shiraz (Jafari et al., 1996), Tehran (Meshgi et al., 2001), Tabriz (Meshgi et al., 2002), Tonekabon (Ranjbar et al., 2005), Golestan (Ranjbar et al., 2006) and Khozestan provinces (Razmi, 1999).

There is a lack of information about filariasia in the urban area of Urmia in North West of Iran.  In this study, filariosis was indicated in urban dogs of Urmia with two diagnostic methods of the modified knot and rapid antigen tests.

Materials and Methods

One hundred dogs from different parts of Urmia were entered to this study. After the recording of necessary information including age, sex, and breed of the dogs, the blood samples (5 ml) were taken from cephalic or lateral saphenous veins for modified knot test and rapid antigen test.

Modified knot method

The blood samples were mixed with 9 ml formaldehyde 2% and were shacked well for RBC hemolysis. In the laboratory, the samples were centrifuged for 5 minutes in 1500 rpm. Methylene blue stain was added and the mixture was examined for the presence of microfilaria with photomicroscope. The differential diagnosis between microfilaria of D. immitis and Dipetalonem recaonditum was done regarding the morphological criteria (Eslami et al., 2004).

Rapid test                                                                                                         

Two drops of not coagulated blood, serum or plasma of dogs were added to a concerned place of Heart worm disease kit (manufactured by Bionote, Australia). The result was read after 5-10 minutes.

Results

The results showed that 3 (3 %) of the total 100 urban dogs were infected with D immitis. Blood microfilaria (D. immitis) was observed in 3 samples by modified knot method (Fig. 1). None of the dogs showed positive results based on rapid antigen test kit and all samples were negative with this test (Fig. 2).

Fig 1. Percentage of healthy and D. immitis-infected dogs in Urmia distinct.

Fig. 2. Rapid antigen test for detection of D. immitis infection in dogs in Urmia distinct.

The dogs were from different breeds including Sarabi (Persian mastiff), German shepherd, Terrier and Boxer (Table1). Regarding epidemiology of the D. immitis, dogs with age of above 1 year old entered to the study. Three age groups of the dogs including 1-3, 3-5 and >5 years old were compared by both modified knot method and rapid ELISA kit. There was one infected dog to D. Immitis in each age group. No significant difference in dirofilaria infection was found among different age groups (P> 0.05). Concerning the gender of the dogs, two males and 1 female dog were infected by D. Immitis. There was no significant difference in dirofilaria infection between male and female dogs (P> 0.05). (Table 2).

Table1. Frequency of different breeds in urban dogs of Urmia

Table 2. The seroprevalence of dirofilariasis in urban dogs of Urmia with different ages and sexes.

Discussion

Studies in the different regions of Iran showed that the infection to D. immitis or heart worm is distributed in many areas of the country. The extensive climatic distribution of the infection to the above-mentioned nematode is as follows: Ardabil (34.6 %) (Bokai et al., 1998), Shiraz (9.5 %) (Jafari et al., 1996), Tabriz (4.8%) (Meshgi et al., 2002), Tehran (1.4 %) (Meshgi et al., 2001). This nematode was also reported in wild carnivores in Khozestan province (Eslami, 1998). Accordingly, different distribution of D. immitis infection has been world-widely reported including Taiwan (13.8 %) (Fon et al., 2001), Mexico (7.3 %- 7.5 %) (Reifur et al., 2004), Canada (0.04%-0.19%) (Klotins et al., 2000).

The present study showed that the infection rate of D. immitis in urban dogs of Urmia is 3%. This is accordance with the results of some other studies in different regions of Iran and it is almost in the ranges reported from Tabriz (4.8%) (Meshgi et al., 2002) and Tehran (1.4 %) (Meshgi et al., 2001).  

According the present study, the rapid antigen test is less sensitive than the modified knot test. As the modified knot test was able to identify three positive cases, the rapid antigen test was not able to detect any antibody. It can happen probably due to little number of D. immitis in the primary phases of the disease. This finding is not compatible with a study in South America which has been shown that the rapid antigen test is as sensitive as the modified knot test (Vezzani et al., 2007).

The statistical analysis did not show any significant relationship between the infection to heart worm and sex, age and breed of animals. These finding were also in line with those of other studies (Ranjbar et al., 2005), but the study in Brazil showing that the infection in the male dogs (89.7%) was higher than in the females (Souza et al., 1997). A study in Spain reported that the highest rate of the infection to D. immitis was observed in the age range of 6 years upward (Montoya et al., 2006).

In comparison with a study in shepherd dogs of Urmia (Javadi et al., 2011), which showed the infection rate of 24/8%, a remarkable difference is noticed. The reason may be related to the different living style in dogs living in urban area, which has lesser contact with intermediated hosts.

In conclusion, although the rapid antigen test is rapid, easy and more feasible in practice, it should be accompanied by another test such as the modified knot test.