Dipetalonema evansi Infection in Camels of Iran’s Central Area

MATERIALS AND METHODS

The research was carried out in arid areas of Iran. Three central provinces of Yazd, Isfahan and Kerman were selected. These regions are adjacent to the Iran’s central desert. One hundred and fifty apparently healthy dromedaries of both sexes and different ages were selected randomly and jugular vein blood was withdrawn, mixed with EDTA and kept in refrigerator (2-8°C) until examination before 24 h. Diagnosis was made by examination of wet or stained thin blood films or by concentration of microfilaria by Knott technique to detect Dipetalonema evansi microfilariae (Anonymous, 1977; Soulsby, 1982).

In a second study on 144 dromedaries (65 males and 79 females) slaughtered in Yazd, Najaf Abad and Rafsanjan abattoirs, we carried-out careful clinical examination before slaughter and also gross inspection for presence of D. evansi after slaughter. Specimens of blood, testis, epididymis, spermatic cord, trachea, bronchi, lung and also heart of them were obtained. Blood smears were prepared similar to the above-mentioned study. Infected and suspicious organs were transferred into Normal Saline Solution (NSS) for washing, centrifugation and subsequent collection. The recovered helminthes were examined by light microscopy in fresh state and also as fixed in 70% ethanol followed by lactophenol treatment, after which they were counted and measured. Identification and measurements of this worm were conducted according to keys described by Soulsby (1982). Tissue samples of infected testes were fixed in 10% neutral buffered formalin, dehydrated in graded ethanol, embedded in paraffin, sectioned at 5 μm thicknesses, stained with hematoxylin and eosin and studied with a routine light microscope by a parasitologist and a pathologist.

The obtained data were analyzed using SPSS software for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA). To compare relative frequency of infection between different age groups of camels, Chi-square test was used. Differences were considered significant when p<0.05.

RESULTS AND DISCUSSION

Sheathed microfilariae were found in peripheral blood smears of 38 (12.92%) out of 294 tested camels. D. evansi adult worms were recovered from at least one of the below organs in 20 (13.89%) camels: epididymis, testis, spermatic veins, heart and lungs (Table 1). The maximum adult nematode infection rate was seen in testes. The overall infection rate of male camels with either microfilariae or adult worms was significantly higher than females (p = 0.0001). In two tested males, simultaneous adult D. evansi worms’ infection in three major organs of testis, heart and lungs was observed.

Table 1:	Distribution of affected organs in two sexes

D. evansi mature nematodes were recovered from either of the arteries of the spermatic cord, testicle, epididymis, lungs and heart of infected camels. The worms recovered from organs had different lengths, females being larger (14.8-21 cm) than males (8.5-10.5 cm). The largest and smallest spicules were 550-700 and 160-180 μm long, respectively. Microfilaria measured 200-300 μm in length.

The main pathologic findings in the studied tissues included sections of parasite, severe acute and chronic inflammation, fibrosis and atrophy. More detailed microscopic evaluation of the testis showed severe atrophy of the seminiferous tubules, thickening of their basement membrane, Sertoli cell-only syndrome (total germ cell aplasia) and sloughing of germ cells in some tubules. Only rare spermatozoa could be found in most of the affected testes. Usually, the interstitial Leydig cells were preserved. The surrounding tissues displayed cross sections of the worms, profound infiltration of eosinophils, lymphocytes, some histiocytes and some neutrophils. Peripheral to this severe inflammation, there was usually marked fibrosis and disarrangement of the tissues. Blood vessels in the affected regions showed prominent endothelial cells. There were some fresh and/or old hemorrhagic areas. Granuloma formation was occasionally detected. In the case of long-standing tissue invasions by the worm, hyalinization or calcification was found as a clue to its chronicity. Spermatic cord and epididymis also manifested similar pathologic findings (Fig. 1-3).

The prevalence rate of Dipetalonema evansi microfilaria in blood in the present study was 12.92%. Researchers from different geographical regions of Iran have reported various infection rates of camels with microfilarial form of this worm. Fard et al. (2011) and Borji et al. (2009) reported lower rates down to 2-3 and 5.34%, respectively.

Fig. 1:	D. evansi adult parasites in the testicular tissue (Hematoxyline and Eosin stainingx100)

Fig. 2:	Chronic granulomatous inflammation with multinucleated giant cells around the worms (Hematoxyline and Eosin stainingx100)

Fig. 3:	Sertoli cell-only syndrome in the affected camel testis (Hematoxyline and Eosin stainingx400)

However, higher rates of 20.7, 28.1 and 46.7% are reported from camels in other parts of the country (Rahbari and Bazargani, 1995; Mowlavi et al., 1997; Oryan et al., 2008). The difference in reported rates of infection may be due to the difference in management systems of the dromedaries.

Although the rate of circulating microfilarial infection in this study was higher among female than male dromedaries (20.16 vs. 7.64%), but it was not statistically significant. Similarly (Borji et al., 2009; Fard et al., 2011) found that infection rate of D. evansi was not significantly different between male and female camels.

In addition, the prevalence rate was highest in animals aged below 5 years. Similarly, Rahbari and Bazargani (1995) observed a significant inverse relationship between host age and prevalence of infection. It may be due to decrease in susceptibility with advancing age (Muhammad and Athar, 2000). In contrast, (Borji et al., 2009; Fard et al., 2011) reported that the infection rate increased with age, although no significant relationship between the rate of infection with D. evansi and age was observed.

In our study, 15.28% of tested camels showed adult worms of D. evansi at necropsy. Infection was higher in males (17/22) than females (5/22). In a study by Fard et al. (2011) on 309 camels slaughtered at Kerman slaughterhouse in Iran, 49 (15.85%) of them from different ages and both sexes were infected with adult forms of D. evansi in either of their testicle, epididymis, spermatic cord, or lungs. Most of the other researchers in Iran have studied infection in one sex (males) or one organ and reported various rates of adult worm infection. Oryan et al. (2008) and Borji et al. (2009) studied only male dromedaries and reported that 20.7 and 5.34% of their tested camels were infected with adult worms, respectively. Mowlavi et al. (1997) found that half of the Iranian male dromedaries (50%) in Najaf Abad and Yazd abattoirs harbor testicular D. evansi. Moghaddar et al. (1992) however, reported adult worms in the lungs of 17.5% of 40 tested camels in Shiraz province, Iran.

Histopathologic findings in the present study are almost in agreement with previous studies.

Results of this study showed that dromedaries in central part of Iran frequently suffer from low grade infections with D. evansi, which indicates the necessity of more research on outstanding reproduction, tissue migration, vectors and host-parasite interactions of this filarial nematode parasite.

ACKNOWLEDGMENT

The authors would like to thank Tehran University of Medical Sciences for financial supports and Ms. Dr. Gilda Eslami for proofreading of the manuscript. We also would like to acknowledge Mr. Mohammad Javad Kazemi and Mrs. Zahra Asayesh for their assistance.