Emerging Haemosporidian Infections in Village Chickens ( Gallus gallus domesticus ) in Yobe State, Nigeria

: In some parts of Nigeria, studies have revealed avian haemosporidian infections in village chickens, some of which are considered emerging parasitic infections because chickens are unnatural hosts. Infections may have occurred as a result of accidental cross-infection via infected haemophagus arthropod vectors. This study was carried out to determine the emerging avian haemosporidian infections in Village chickens in Yobe State, Nigeria using microscopy. Blood samples were collected from 2100 Village chickens in 7 Local Government Areas of Yobe State. Microscopy revealed an overall prevalence of 13.8% (290/2100) for avian haemosporidians comprising of Plasmodium with 13.9% (198/2100) and Haemoproteus 2.4% (55/2100) as single infections. Mixed infection of Plasmodium and Haemoproteus had a prevalence of 1.8% (37/1820). Male (9.9%) had a higher prevalence of avian haemosporidian infections than female chickens (4.0%). Adults (10.4%) had a higher prevalence than growers chickens (3.4%), and prevalence was higher in the rainy (9.3%) than the dry (4.5%) season of the study period. Detection of Haemoproteus specie in chickens in Yobe State is considered an emergent infection because chickens are not natural host to the parasite. It is therefore recommended that Village chicken producers should be educated on the risk factors and impacts of avian haemosporidian on bird productivity through frequent agricultural extension services. More research is needed to assess the frequency of avian haemosporidian infections in other poultry species in Nigeria, in order to better understand the disease's epidemiology and develop effective management and prevention strategies.


Introduction
Poultry is raised by a large number of rural people around the world, and it serves an important economic role in delivering rich animal protein (meat and eggs) and alleviating poverty [28]. Small-scale chicken production operations in impoverished nations involve breeding a few birds that are not provided with contemporary housing facilities for roosting at night, scavenge around households, receive no medical attention, and are not routinely vaccinated [38].
Infectious poultry diseases such as viral, bacterial, protozoan, and parasitic diseases are a major barrier to a successful village chicken production system in developing countries [7,15,36,43], but avian haemosporidian infections and their consequences in village chickens have received little attention. Village chickens are typically reared under the extensive management systems in most parts of Nigeria [26]. They have access to the environment where they scavenge for food even on unhygienic garbage dumps and near unhygienic pool of water [2]. Village chickens are more likely to be subjected to bites from many types of haemophagus arthropod vectors that may host blood parasites compared to chickens raised in screened pens or restricted to intensive management systems [15,17,35]. Although several published research has identified blood parasites primarily in wild and other birds in tropical areas worldwide, recent studies have found emerging haemoparasite infections in (Gallus gallus domesticus) in Yobe State, Nigeria poultry species including village chickens [1,27,39]. Plasmodium, Leucocytozoon, Haemoproteus, Microfilaria, Aegyptinella, Fallisia and Trypanosoma species are genera of haemoparasites recorded in chickens around the world [3,6,41]. Avian haemosporidian is an emerging parasite infection in domesticated birds, and there has been an increase in research revealing avian haemosporidian infections in domesticated birds in some parts of Nigeria [9, 14, 25, [27], raising concerns. However, because there is no published data on the prevalence of avian haemosporidian infections in village chickens in Yobe State, Nigeria, a study on the prevalence of haemosporidian infections in village chickens is needed to develop strategies for effective control programs and to improve the safety and productivity of village chickens in the study area.

Study Area
Yobe State is located in northeastern Nigeria, with Damaturu as its capital and Potiskum ( Figure 1) as its main city. The state shares borders with four states: Bauchi, Borno, Gombe, and Jigawa, as well as the Diffa and Zinder regions of Niger to the north. Because the state is primarily located in the dry savanna zone, the climate is hot and dry for the majority of the year.

Study Design and Study Population
After getting consent from their owners, a total of 2100 village chickens were sampled from families that rear several poultry species as part of a cross-sectional study that used non-probability sampling procedures.

Study Period and Blood Sample Collection
Blood samples and other data collections were carried out from September 2019 to November 2020. Blood samples were aseptically collected from each sampled village chicken via the wing vein (venipuncture) using sterile 5ml syringes and 23 gauge needles. Blood samples were immediately dispensed into sample bottles containing EDTA as anticoagulant and transported in cool flasks to the Department of Veterinary Parasitology and Entomology Research laboratory for processing and microscopy examination.

Blood Smear Preparation
Thin blood and buffy coat smears were made on a clean dry slide according to standard protocol as described by Mello et al. [18], smears were allowed to air dry for a few minutes, then fixed in absolute methanol, and then allowed to air dry again, before properly labeling each slide.

Microscopic Detection of Avian Haemosporidian
The slides were stained with Giemsa stain (pH 7.2), raised with distilled water, and allowed to air dry according to the standard procedures described by Ribeiro et al. [31]. Stained blood smears were later viewed under a light Olympus ® (Japan) microscope first at low magnification (40x), and then at high magnification (100x) oil immersion objective for the presence of intracellular or extracellular blood parasites. The haemosporidian schizonts, gametocytes, and trophozoites were examined and identified based on morphology as previously described by Valkiûnas [41].

Data Analysis
GraphPad Prism software (GraphPad Inc., San Diego, CA) was used to analyze the data. Prevalence rates were calculated as percentages of proportions, the Chi-squared test was used to compare categorical variables (age, sex, and season), and the relative risk calculated using the GraphPad Prism software. The lower and upper limits of the 95% confidence interval for a proportion were calculated using the procedures provided by Newcombe [22]. Differences were considered significant for p-values equal to or less than 0.05.

Discussion
The present study showed 13.8% overall prevalence of haemosporidian infections in village chickens from Yobe State, Nigeria. This is the first recorded avian haemosporidian prevalence in the study area which makes it an emergent infection in the poultry sector in this part of Nigeria. Our result exceeds that of George et al. [ [9], and 23.2% in Makurdi, Benue State [25]. The disparities in reported prevalence rates of haemosporidian infections in village chickens from various studies could be attributable to sample sizes, geographical locations, as well as the availability of arthropod vectors and season, which could influence vector breeding. Furthermore, higher prevalence rates of avian haemoparasites in village chickens have been documented in some parts of Africa, including 43.4% in Ethiopia [4], 79.2% in Kenya [32], 71.0% and 79.1% in Malawi by Njunga [23] and Lutz et al. [16] respectively, 61.9% in Uganda [42], 35.0% in Ghana [30], 32.0% in Zimbabwe [29]. These studies demonstrated an increasing evidence of avian haemosporidian in scavenging village chickens from diverse locations of Africa.
The high prevalence of avian haemosporidian recorded in village chickens in Damaturu, Potiskum, and Fika Local Government Areas could indicate that these LGAs have an abundance of arthropod vectors capable of transferring haemosporidian infections to village chickens. These LGAs are the state's largest urban center, with an abundance of poor drainage and open sewers that could create an ideal environment for the reproduction of various arthropods, particularly mosquitoes. Moreover, village chickens' vulnerability to haemosporidian infections in these areas may also be exacerbated by their scavenging habit.
Two haemosporidian taxa, Plasmodium and Haemoproteus, were found to be prevalent in this study, indicating the availability of their appropriate vectors in the studied area.
Gimba et al. [6], Nakayima et al. [19] and Nourani et al. [24] have all reported these two avian haemosporidians, indicating that they are the most common in village chickens and have a global distribution. The present study also discovered mixed Plasmodium and Haemoproteus infections, which is consistent with previous findings [6,10,16,20,21,25].

Microfilaria,
Leucocytozoon, Aegyptianella, and Trypanosoma were not found microscopically in any of the samples examined in this study, despite reports by Etisa et al. [4] and Opara et al. [27] that these avian haemosporidian and haemoparasites were found in village chickens.
Plasmodium species (9.4%) was the most common haemosporidian in village hens, confirming the findings of other researchers who found Plasmodium species to be the most common haemosporidian infection in scavenging birds [4,6,8,19,21,32,37]. However, the Plasmodium prevalence rate in this study is lower than 11.4% in Maiduguri [11], 12.0% in Sokoto [40], 33.3% in Owerri [27], and 32.0% in Ibadan [33] of Borno, Sokoto, Imo, and Oyo States respective in Nigeria. Variations in reported prevalence rates of avian haemosporidian infections in Nigerian village chickens could be related to variances in management and husbandry practices, as well as the availability of appropriate vectors, environmental and climatic conditions. The prevalence of a single Haemoproteus infection was found to be 2.6% in this Study. However, Sabuni et al. [31], Gimba et al. [6], and Nath and Bhuiyan [21] from Ethiopia, Selangor, and Bangladesh, respectively, reported lower prevalence rates of 0.9%, 0.8%, and 2.5%. The prevalence of Haemoproteus in village chickens reported in this present study is lower than 23.3% reported in Bangladesh [12], 13.2% in Iraq [10] and 50.9% in Nigeria [14]. This could be due to variances in geographical regions, arthropod vector abundance, and season of sample collection. Village chickens have been reported as an unnatural host to Haemoproteus specie, which has been frequently detected in pigeons and other wild birds. Infection of the chickens by Haemoproteus specie may occur during a blood meal where suitable vectors may visit both host and result in cross-infection from pigeon to chicken, which are commonly reared together in households. In some infected village chickens, a mixed Plasmodium and Haemoproteus species infection was discovered at a prevalence rate of 1.8%. This study agrees with the findings of Naqvi et al. [20], who found mixed Plasmodium and Haemoproteus species infection in scavenging chickens. However, our finding is lower than 47.4% reported by Hasson [10], but higher than 0.5% reported by Nath and Bhuiyan [21].
The results of this study revealed a higher prevalence in males (9.9%) than female (4.0%) village chickens which was statistically significant (p<0.0001), and this could be attributed to the facts that cocks anatomically have larger combs and wattles, that are richly supplied with blood vessels which may attract blood sucking arthropods that transmit haemosporidians to the host bird. Previous studies [14,25,27] have found that male have a greater prevalence of avian haemosporidians than female village chickens in some parts of Nigeria. The findings of our study are also in line with previous reports in Africa and Asia [4,8,32]. It is important to note that other worker have reported higher prevalence of haemoparasites in the hen [10,20,21,34].
The prevalence of avian haemosporidian infections based on age of village chickens in this study has revealed a higher prevalence in adults (10.4%) compared to the growers (3.4%) which was statistically significant (p<0.0001). This might be attributed to the behaviour of adult village chickens which roam far long distance to scavenge for food on or near unhygienic environments and travel far in search of mates, and this predisposes them to higher risks of contact with arthropods vectors, while the growers are usually found near their roosting areas. Also, adult chickens have more prominent and developed combs and wattles which are well vascularized and provide predilection sites for arthropods vectors. This finding concurs with the findings of Abdul Momin et al. [1] in Bangladesh and Etisa et al. [4] in Ethiopia who also reported significantly (p<0.05) higher prevalence of haemoparasites infection in adult chickens compared to growers. However, this finding contradicts that of Sabuni et al. [32] in Kenya and Naqvi et al. [20] in Pakistan who reported a relatively higher prevalence of avian haemosporidian infections in growers compared to adult chickens, but the difference was not statistically significant (p > 0.05).
This present study has revealed a significantly (p<0.0001) higher prevalence of haemoparasites during the rainy season (9.3%), which is the season that provides favourable conditions such as temperature and humidity that allow breeding of most arthropod vectors such as mosquitoes and other biting flies. The prevalence recorded in this study agrees with the findings of Igbokwe et al. [11] who also reported a high prevalence of avian haemoparasites during the rainy season in Maiduguri, Nigeria, but contrasted with Nath and Bhuiyan [21] who reported higher prevalence of haemoparasites in dry season.

Conclusion and Recommendations
In Yobe State, Haemosporidian parasites of Village chickens such as Plasmodium and Haemoproteus species are prevalent (13.8%; 95% CI=12.4%-15.4%). The prevalence of haemosporidian infections were significantly higher in male, adults chickens and higher in the rainy season of the study period. Plasmodium (9.4%) was discovered to be the most prevalent haemosporidian in village hens in Yobe State, and Haemoproteus species discovery in chickens is considered an emerging infection because chickens are not Haemoproteus species' native hosts. Village chicken farmers should be educated about the risk factors and effects of haemosporidian infection on bird productivity by regular extension services, with an emphasis on control and prevention methods. To better understand the epidemiology of the disease, more research should be done to assess the prevalence of avian haemosporidian in other avian species in the study area. To characterize the Haemosporidian species infecting village chickens in Yobe State, more advanced molecular approaches should be used.

Conflicts of Interest
The authors declare that they have no competing interests.