Identification of Blood Protozoa in See-See Partridges (Ammoperdix Griseogularis) in Garmian Area/Kurdistan Region-Iraq

ا ا نع فشكل و يف مدلا يلا لجح عون درفصلا ( Ammoperdix griseogularis ميلقإ/نايمرك ةقطنم يف )

Avian haemosporidians usually cause losing weight, lowering productivity especially egglaying in birds and lower development rates, in addition to the direct effect caused by anemia, and destruction of cell body and allergic response might occur (4). Few studies also have suggested that these types of haemosporidians infections can cause reduced speed and strength in flight, poor appetite, anemia, air sacculitis, and arthritis in hosts (5).
Iraq has rather a wide range of geological diversity, from the peaks of Kurdistan Mountains in the north to the deserts and semi-deserts in the west and the great Mesopotamian marshes in the south; these share in the enormous biological and avian diversity. See-see partridge (Ammoperdix griseogularis) is a game bird in the pheasant family Phasianidae of the order Galliformes (6), has a huge distribution range, from southeast Turkey through Syria and Iraq to Iran and Pakistan (7,8), it is considered as one of the recorded birds in the checklist of Iraqi birds, where widespread breeding resident in hills and mountains in northern and eastern areas of this country (9). See-see partridge takes a great variety of seeds and some insect food; usually, it is seen in pairs or, at the most, in flocks of two to four birds. However, flocks have also been seen of more than fifty birds. When disturbed, the partridge prefers to walk rather than fly, but if needed it flies a short distance on rounded wings (10).
To date, Haematozoa of Iraqi birds are rather partially known based on many published studies on the different bird species usually comprising domestic chickens, pigeons, doves, ducks, geese, turkeys, passerines, chukars, partridges, quails, resident, wild birds, and migratory avifauna and some others, which were mostly infected with one or more species of Haemoproteus, Plasmodium and Leucocytozoon (5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Even though much more researches had been carried out in various species of wild and domestic birds in Iraq by Mohammad et al. (16) and Shamsuddin and Mohammad (27) who were the first authors studied on see-see partridge birds in terms of Haematozoan parasites in this country the former found Haemoproteus and Plasmodium spp. with the rates of infection 30.9% and 2.4% respectively, and the latter recorded Haemoproteus spp. for instance with the rate 75%, after that, no work had been studied on this aspect in these birds. So far due to a lack of knowledge concerning the distribution, prevalence, and identification of haemosporidian parasites, especially in see-see partridge in the Kurdistan region-Iraq, thus the objective of the current study was originally designed and conducted to isolate and detect various blood protozoan parasites in this type of bird within a given geographical or ecological unit in Garmian area/Iraqi Kurdistan region relating their prevalence of infection in these partridges.
After the partridges being captured, they were transferred to the parasitological laboratory of Kalar Technical Institute /Sulaimani Polytechnic University and kept them alive in separate cages, then tagged with a leg ring to avoid duplicate sampling.
. Figure 1. Garmian Area map, Geographical map of the study area around the Garmian area (28).

Blood samples collection and preparation of Giemsa-staining blood smears
After the wing vein of each sampled bird was disinfected by 70% ethyl alcohol, a small amount of blood was oozed by puncturing the vein with a sterilized disposable lancet (29), then the blood smears were prepared as suggested by Sriraman (30) as follows: A small drop of blood was placed on the end of a clean glass slide, then the blood drop was spread by the end of another glass slide which was placed in front of the drop at an angle of approximately 30 degrees and pushed back until it just touched the drop, immediately after that, the spreader slide was pulled forward smoothly and quickly at the same angle, the blood smears were air-dried by waving the slide in the air, then all slides were labeled with specific numbers and kept in box slides until the time of staining.
Staining of blood smears for the detection of haemosporidian parasites The blood smears were fixed in absolute methanol for 3 minutes, after they were air dried, they immersed in working Giemsa staining solution 1:10 at pH 7-7.2 for 30 minutes, directly after staining, then were washed in distilled water, and air dried finally, examined microscopically using an Olympus BX43 light microscope; at least 100 fields were examined under 400X and oil immersion (1000×) magnification (30), morphological identification of detected parasites and red blood cells was performed following the taxonomic keys of (24,31,32).

Results and discussions
An examination of the blood smears indicated the existence of different intracellular stages of haematozoan parasites consistent with Plasmodium spp. and Haemoproteus spp. (Fig. 2: A, B, C, and 4). Out of 63 microscopic examination of the stained thin blood smears from different ages and sexes of healthy adult seesee partridges 68.58% was an overall infection rate in which Plasmodium parasites were the more frequent blood parasite detected in the present study, thus it was clear that 32 birds were found infected with Plasmodium spp. with an overall prevalence rate of 50.79% (Table 1), these protozoa were characterized by intraerythrocytic halter-like gametocytes together with signet ring-shaped meronts (Fig. 2 A) during the microscopic examination of the Giemsa Vol. 14 Issue:1, (2021) ISSN: P-1999:6527 E-2707:0603 stained blood smears of infected bird with Plasmodium parasites, in which morphological alterations and nuclear displacement were noticed in some erythrocytes because of the presence of these gametocytes and meronts ( Fig. 2: A, B and C). The findings as well as showed that 8 examined birds were found infected with Haemoproteus spp. parasites, with an overall infection rate of 12.69% (Table 1). The microscopic examination of blood smears of the examined birds infected with Haemoproteus parasites revealed the presence of intra-erythrocytic gametocytes occupying about one-half of the erythrocyte cytoplasm with little or no displacement of the host cell nuclei; they surround the nuclei either partially or completely, also, brown pigment granules from digestion of host hemoglobin (hemozoin pigment granules) are evident within the infected erythrocytes (Fig. 3). It is worthy to mention that this study recorded a new bird host which is see-see partridge for the haematozoan parasites such as haemoproteus spp. and plasmodium spp. Three positive cases were revealed mixed infected from overall examined blood smear samples with Plasmodium and Haemoproteus parasites with an infection rate of 4.76%. As shown in Table 1 see-see partridges, and this was the aim of the current study. As much as we know, this study is the first attempt to detect and investigate the infection rates of haemosporidian in this type of bird in the Iraqi Kurdistan region.
The interpretation of this result is somehow similar to those shown by (33,34) in which; some avian species appeared to be more susceptible than others. For instance, ducks, geese, and swans are commonly infected with species of Haemoproteus, Leucocytozoon, and Plasmodium, and more than 75% of waterfowl species were hosts for one or more of these parasites, it is obvious that pigeon and dove species have similarly high rates of infection, but genera of other families, such as migratory shorebirds, are less frequently parasitized.
From the results, out of the 63 see-see partridges involved in this study, only 43 of them were found infected with haemosporidian parasites. As an Iraqi area, Garmian is a subtropical region accordingly the infection rate (68.25%) reported in this study appears to be variable in comparison with infection rates reported in birds in the nearby tropical and subtropical regions (35,36) and in subtropical regions around the world (37).
So far, (16,27,38) (Table 1), often these variations might be influenced by many real factors related to the variation in the incidence or prevalence like health status of bird, sex, age, feeding and feeding habitats, season, climatic variables such as temperature and the presence of transmitters (39).
As well as the high infection rate (68.25%) of haemosporidian parasites in the present study might be stemmed from different related reasons as mentioned above. The first reason is the time of sampling. The blood samples of see-see partridge were collected during the period extended from November 19thof 2018 to March 15th of 2019. This period was almost wet and thus the proliferation of insect vectors could be largely activated, so this finding contrasts with many observational reports that pointed out a low prevalence of haemosporidian parasites in birds sparsely exposed to vectors (30,40). In fact Vol. Generally, the pathogenesis of parasitic diseases depends upon many factors such as the age of the host species and its health status, site of infection, and numbers of parasites (44), however, no pathological signs were observed in the partridges examined in this work.

Conclusions
It was concluded from the present study that seesee partridges might be infected with Plasmodium and Haemoproteus blood parasites in Garmian area/ Kurdistan Region-Iraq. The results of the present study explored for the first time the existence of the Plasmodium spp. and Haemoproteus spp. in the partridges in this area. Really the various geographical areas with different climatic changes are the most powerful impact on the rates of infection with these haematozoa. In this work, the rate with Haemoproteus spp. parasite was lower in comparison with plasmodium one. As it was recorded, the birds were really susceptible to single and/or mixed haematozoan infection with various rates, and this might become a source of infection to domestic and wild birds in our region through the distribution of vectors and lead to an economic loss of poultry in our country.