Molecular Detection of Avian Malaria (Plasmodium gallinaceum) in Local Domesticated Breed Chickens (Gallus gallus domesticus) in Baghdad

This study was conducted to investigate the prevalence of avian malaria (Plasmodium gallinaceum) in the local domesticated breed chickens (Gallus gallus domesticus) that were purchased from the local markets in Baghdad city, using 100 blood samples which were collected from the wing vein, and kept in EDTA-K2 tubes for conventional PCR analysis during the period extended from 1 /10 / 2018 till 31/ 3 / 2019. Total infection rate was 18% (18/100), which were divided into males 20.00% and in females 16.00%. The eight isolates were recorded in the GenBank under accession numbers ID: MN082405.1, MN082406.1, MN082407.1, MN082408.1, MN082409.1, MN082410.1, MN082411.1, and MN082412.1 with identity 99.20 99.87% and with other isolates (United Kingdom and USA) 99.34 99.88 %. In conclusion, Plasmodium gallinaceum may have a moderate spread in local domesticated breed chicken at Baghdad. H *Correspondence: duraali72@gmail.com Received: 23 March 2020 Accepted: 28 Jun 2020 Published: 28 December 2020 DOI: https://doi.org/10.30539/ijvm.v44i(E0).1025 This article is an open access distributed under the terms and conditions of the Creative Common Attribution License (CC BY 4.0)


INTRODUCTION
aemosporidian infections are protozoa parasites that causing diseases by a group of parasites that infect mammals, reptiles, birds, and amphibians (1). The diseases are induced by an unknown number of haemozoin species (2), and most often only Plasmodium spp. are referred to as avian malaria parasites (3), that as a haemoparasitic infectious (4)(5)(6). The great majority of described avian Plasmodium species were reported only in birds that live in tropical and subtropical countries and which have adapted for transmission globally and have become cosmopolitan (7). It may be severe mortal in zoos and for domestic birds (8). It is referred that P. gallinaceum, P. juxtanuclear and P. duraeappeared to be the most dangerous for poultry causing up to 90% mortality (9). Life cycle of these parasites need two hosts undergo in vectors invertebrate host, sexual reproduction (10), Culex mosquitoes (11) that only develop in females (1, 12) and in vertebrate host that is asexually (13).

A B S T R A C T
This study was conducted to investigate the prevalence of avian malaria (Plasmodium gallinaceum) in the local domesticated breed chickens (Gallus gallus domesticus) that were purchased from the local markets in Baghdad city, using 100 blood samples which were collected from the wing vein, and kept in EDTA-K2 tubes for conventional PCR analysis during the period extended from 1 /10 / 2018 till 31/ 3 / 2019. Total infection rate was 18% (18/100), which were divided into males 20.00% and in females 16 There are different diagnostic methods used for the detection of the haemosporidian parasites. Traditional method detects the infection by microscopic examination of blood smears only, while the development of new molecular technology such as polymerase chain reaction (PCR) has made detection for these parasites more reliable and faster (4) as it can indicate the high diversity of parasites (14,15) reaching to the species level by sequencing the amplified DNA (16).
Erythrocyte invasion by Plasmodium parasites requires apical membrane antigen-1 (AMA-1) and rhoptry neck protein 2 (RON2) interactions. These proteins of P. gallinaceum are evolutionarily and structurally conserved and the suggesting may play essential roles for invading host erythrocytes. In addition, a high level of genetic variation was found in ama-1 of avian Plasmodium species and these findings have implications for investigating (17).
Due to the important effects of avian malaria on economic loss and mortalities, and their prevalence is not well known by using molecular technique (PCR) in local breed domestic chickens in Baghdad city, this study was conducted.

Animals, Areas and Periods of the Study
All procedures conducted in this study was reviewed and approved by the scientific committee in the College of Veterinary Medicine, University of Baghdad in accordance with the ethical standards of animal welfare.

Blood Samples
About 1-2 mL of wing blood samples were collected from each bird by a sterile syringe and kept in EDTA (Ethylene diamine tetra acetic acid, K2) tubes (18) and stored in -20 °C until for PCR analysis.

DNA Extraction
Genomic DNA from the whole 100 blood samples was extracted using a G-spin DNA extraction kit (Cat. No.17045, Life Science, Korea). Concentration and purity (absorbance at 260/280 nm) of extracted DNA were checked using a Nanodrop spectrophotometer (Thermo Scientific, USA).

Primers
Primers used according to Lauron et al. (17) for the partial amplification (875 bp) of the ama-1 gene are shown in Table 1. The lyophilized primers were dissolved in nuclease free water to give a final concentration of 100 pml/µL as a stock solution and kept at -20 °C. Then, a concentration of 10 pmol/µL was made as working primers, in which 10 µL of the stock solution was added to 90 µL of deionized distilled water (ddH2O) to reach a final volume of 100 µL (Integrated DNA Technologies Company).

PCR
The PCR reaction mixture involved the use of iNtRONsMaxime PCR PerMix kit. The components of the kit included: i-Taq DNA Polymerase 5 U/µL, dNTP mixture 2.5 mM, reaction buffer 1x, gel loading buffer1x. The PCR working solution in a final volume of 25 µL included: 5 µL of Taq PCR PreMix, 10 pmol/µL (1 µL) of each forward and reverse primers, 1.5 µL of the DNA templates as well as 16.5 µL of nuclease-free water.

Sequencing of Gene and Sequence Arrangement
The PCR amplicons were sent for sequence (8 samples) to Macrogen company (Korea). The sequencing was performed by National Instrumentation Center for Environmental Management, biotechnology lab using DNA sequencer 3730XL (Applied Biosystem). Homology search was conducted using Basic Local Alignment Search Tool (BLAST-NCBI) program, which is available at the National Center, Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). The phylogenetic tree diagrammatic was done by molecular evolutionary genetic analysis (MEGA) software version 6.0Sequences (10,21).

Statistical Analysis
Chi-square was used to determine the effect of sex in the infection rate at a significant (P< 0.05) difference level (20).

Phylogenetic Analysis
The phylogenetic relationship of the eight local isolates of Plasmodium gallinaceum was between 99.20-99.87% and between these isolates and the world isolates between 99.34-99.88% (Figure 2

DISCUSSION
Haemosporidian infections are group of diseases caused by protozoan parasites that infect different animals including birds, their community, and may reflect a largescale of geographical variations (1, 22). Eggs and meat of the family use are exposed to parasitic diseases such as haemosporidians (23). Shadan (24) observed that Plasmodium spp. was the most prevalent haemoparasites 52.63% (70/133), Flayyih (25) recorded an infection was 9.86% in turkey. These infection rates were differered from the present result (18%) in which it was less or higher and that may be due factors related to the sampling effort, location, poultry breed, immune status, habitat type, season, the abundance of arthropod vectors responsible for transmitting the parasites (26), behavior and diet (27). The great majority of described avian Plasmodium species were reported only in birds that live in tropical and subtropical countries or in Holarctic migrants wintering in the same regions, indicating that transmission of these pathogens occur mainly in countries with warm climates. The malaria parasites, which have adapted for transmission globally and have become cosmopolitan; Molecular diagnostics using general primers (the main diagnostic tool currently used in wild life malariology) is often insensitive in distinguishing of avian Plasmodium spp.co-infections, which are common and even predominate in many bird populations (7). In the present study use PCR as molecular markers which are sensitive for distinguishing different parasite species and their lineages, and they are essential for the identification of cryptic Plasmodium species (28 ) and it is more sensitive method for detecting the Plasmodium when compared with conventional methods, and the microscopy diagnosed give 50% false-negative results when confirm by PCR (9), (29). Morphological identification using microscopic examination of blood films remains an important for malaria diagnostics .It is particularly valuable if applied with PCR as based diagnostic tools. During the past 15 years numerous avian Plasmodium parasites were named and described using morphological features of their blood stages and molecular markers for parasite detection were developed in a handful of these descriptions (7).
In conclusion, high infection rate of Plasmodium is found in domesticated local breed chickens. Sex is significantly affected in the prevalence of Plasmodium infections in chickens and the high infection rate is found in males more than females.