Prevalence and Morphological Characterization of Cheilospirura hamulosa, Diesing, 1861 (Nematoda: Acuarioidea), from Partridges in Iran

This study reports data on the prevalence, morphology, and morphometry of the nematode Cheilospirura hamulosa on the basis of light and stereoscopic microscopy and also camera lucida. Specimens were recovered after necropsies of 100 partridges (Alectoris chukar) from Taleqan County in Alborz Province, Iran. The prevalence of C. hamulosa in partridges was of 30% with a mean intensity of 3.9 and range of infection of 1–12. The mean length and width of females were 17.5 ± 2.14 and 0.39 ± 0.04 mm, while those of males were 12.2 ± 0.67 and 0.3 ± 0.06 mm, respectively. The characteristic digitiform tail was observed in females, and the unequal spicules, caudal alae, and ten pairs of caudal papillae were seen in males. The taxonomic characteristic longitudinal cordons and muscular and glandular oesophagus were observed in both sexes. Ratio between cordons and body length in males and females was 1 : 1.33 and 1 : 1.68, respectively. Ratio between long and short spicules in males was 1 : 2.3. The average size of embryonated eggs was 51.25 × 29.5 μm. In the present study, C. hamulosa (Nematoda: Acuarioidea) is recorded for the first time from partridges in Iran. Therefore, the morphological characters described in this study will be useful in the future diagnostic and taxonomic studies of Acuarioidea family.

The C. hamulosa is located under gizzard cuticle mainly in koilin or muscular wall of the host [1,2]. This nematode has indirect life cycle. The grasshoppers (Melanoplus, Oxyanitidula, and Spathosternum parasinifrum), beetles, and weevils are intermediate hosts for C. hamulosa and birds acquire their infections by eating contaminated arthropods containing infective third-stage larvae [1]. Due to its high pathogenicity in poultry, the C. hamulosa has veterinary and public health importance. Cheilospirura hamulosa can cause several complications such as granulomas and nodules that lead to anemia, impotence, and mortality in chickens [2]. Cheilospirura hamulosa may cause zoonosis and has been recovered from a nodule on the conjunctiva of a Filipino farmer [5]. The previous studies in rural areas of Iran indicated that the infection of this worm is common among free-range chickens [6], whereas there is no published data on partridges in Iran. The main goal of the present study

Materials and Methods
The study was conducted in Taleqan County. This area is located in Alborz Province and its height is 1900 m above the sea level. Latitude and longitude of Taleqan are 36 degrees 15 N and 50 degrees 46 E, respectively.
One hundred partridges were collected from Taleqan mountainous region between 2011 and 2013. Every year from the end of September to the beginning of February a license is issued by the Environment Protection Agency (EPA) of Iran for hunting partridges. Every hunter has permission to hunt the maximum of three partridges weekly. For this research, the gizzard of each partridge was removed from the alimentary tract and delivered to the helminthology laboratory of Shahid Beheshti University of Medical Sciences. The gizzards were examined macroscopically and then dissected in a 0.85% NaCl solution (normal saline) to remove cuticle. Worms were visible to the naked eye. The worms were rinsed in normal saline and fixed in Alcohol-Glycerin (70% Alcohol, 50 mL; Glycerin, 50 mL) solution. Nematodes were elucidated with acetic acid and phenol, mounted in Canada balsam. Helminthes were counted and identified under light microscopy (Zeiss, Germany) and stereoscopic microscope (Zeiss, Germany) and traced by camera lucida (Zeiss, Germany). The morphological identification of the nematodes to the species level was done according to methods described by Skrjabin et al. [9].

Prevalence of C. hamulosa.
Out of a total of 100 partridges examined, the prevalence of C. hamulosa was 30% with a mean intensity of 3.9% and range of infection of 1-12. A total of 116 worms were recovered from partridges. Among the recovered helminthes, 60.3% were female and 39.7% male. The specimens of C. hamulosa were found free under the gizzard cuticle, partially or fully burrowed in the walls ( Figure 1). Microscopic description was based on 10 adult worms, five males and five females (Table 1).

Discussion
The nematode C. hamulosa reported herein is reported for the first time from partridges in Iran. Studies on this nematode in Iran only was carried out in native fowls. The partridge (Alectoris chukar) is the most important bird hunted in Iran. In the present study, prevalence of C. hamulosa in the partridge was 30% with a mean intensity of 3.9% and range of infection of 1-12.
The reported prevalence of this nematode by Menezes et al. [2] in Brazil was 14.3% in ring-necked pheasants (Phasianus colchicus) with a mean intensity and range of infection of 1.5, 1-2, respectively. In domestic chickens (Gallus g. domesticus), the prevalence, mean intensity, and range of infection were 26.7%, 4 and 1-12, respectively [2]. A 2-year study in Kashmir, India, on the prevalence of the nematode C. hamulosa in indigenous fowl has shown an overall prevalence of 3.5% (17/478) [10]. The prevalence of C. hamulosa in guinea fowls (Numida meleagris galeata Pallas) from Ghana and chickens in Zimbabwe and Cuba was 37.8%, 46.6%, and 84.6%, respectively [11][12][13]. These results are higher than our finding. The reported prevalence of C. hamulosa on native fowls from Golestan Province in north of Iran was 4% which is significantly lower than our result, because the study population is completely different from our study [6]. The nematodes of the genus Acuaria (Cheilospirura) are parasitic among different families of birds and they are located under the koilin layer usually in the cardiac or pyloric regions [2]. Also in our study the specimens of C. hamulosa were found under the gizzard cuticle, partially or fully burrowed in the walls of the organ (Figure 1).
Most of the reported lengths for male and female C. hamulosa are within the range of 9-14 and 15-25, respectively ( [3,14] and [1,7,8]) (Table 1). In this study, male worms were smaller than the female worms in average body length, overall cordon, glandular oesophagus, and muscular oesophagus length (Table 1). Moreover, in our study, the long spicules were smaller and the length of short spicules was longer compared with previous studies reported by Cram [1,3], Yamaguti [7], and Gomes et al. [8] (see Table 1 for comparison). In our study, average length of male worms was higher than those reported by Gomes et al. [8]. In our study, the female worms were smaller in maximum body length than those described by Cram and Yamaguti [3,7] but still were bigger than those described by Gomes et al. [8]. The female of C. hamulosa in our study had smaller muscular and glandular oesophagus than those reported by Gomes and Yamaguti [7,8], whereas values obtained for the length of tail and egg size were bigger compared to two studies mentioned above (Table 1). We observed 10 pairs of papillae in C. hamulosa as reported by Cram [3] and Gomes et al. [8]; however, in the precloacal region, 2 pairs of papillae were unclear (Figure 2, (B1)-(B3)).
Due to the significant pathogenic effects of these nematodes (Cheilospirura spp.) in poultry and wild bird population and very limited prevalence data of these helminthes in Iran, further study will be needed on different aspects of Acuarioidea family including pathogenesis and their prevalence in other avian species.

Conclusions
In the present study, we report for the first time the isolation and morphological characterization of Cheilospirura hamulosa from partridges in Iran. The morphological characters  described in this study will be useful in the future diagnostic and taxonomic studies of Acuarioidea family.