Cross-sectional seasonal prevalence and relative risk of ectoparasitic infestations of rodents in North Sinai, Egypt

Abstract Background and Aim: Rodents are ubiquitous animals that host ectoparasites and transmit zoonotic diseases. We conducted a cross-sectional study on the seasonal variation, period prevalence (Pp), and relative risk of ectoparasitic infestations in rodents collected in North Sinai, Egypt, from September 2019 to August 2020. Materials and Methods: We captured 380 rodents during the study period. Rodents were euthanized to perform species identification, and 2930 external parasites were collected and identified using light microscopic examination with systemic keys depending on morphological characters. Results: Rattus norvegicus (brown rat), Rattus rattus frugivorus (white-bellied rat), Rattus rattus alexandrines (gray-bellied rat), and Mus musculus domesticus (house mouse) were captured at the highest frequencies during summer (n=186), followed by spring (n=84), fall (n=71), and winter (n=39), with a higher proportion of males captured in all seasons. Analysis of the infestation Pp revealed highly significant increases (p<0.01) in ectoparasites during the winter. Temperature, humidity, and dew point were significantly (p<0.01) correlated with the numbers of captured and infested rodents. Parasitological examinations showed the higher risks of flea (Echidnophaga gallinacea, Xenopsylla cheopis, and Leptopsylla segnis) and lice (Hoplopleura hirsuta, Hoplopleura ocanthopus, Hoplopleura oenomydis, and Polyplax spinulosa) infestations during winter and mite (Laelaps nuttalli, Dermanyssus gallinae, Ornithonyssus bacoti, and Myobia musculi) infestations during summer. Conclusion: We conclude that ectoparasitic infestation prevalence and risk varies with predominating macroclimatic conditions. Strict preventive and biosecurity measures should be applied to combat rodent-related problems.


Introduction
Rodents are widespread nocturnal species, representing approximately 40% of all mammals [1]. Rodents are nearly ubiquitous and well-adapted to terrestrial areas from tundras to deserts, playing important ecological roles through their burrowing activities [2]. Rodents contribute to billions of dollars in property and food resource losses annually and negatively impact human and animal health [3]. Rodents are carriers or reservoirs for various viruses, bacteria, rickettsia, and helminths, responsible for transmitting many zoonotic and infectious diseases [4]. These diseases can be transmitted through direct exposure to rodent secretions and excretions or indirect exposure pathways through ectoparasites [5]. Rats are commensal, meaning they live in close proximity and "share tables" with humans; they are known for their adaptability to residential areas and hosting a wide range of ectoparasites [6,7].
Ectoparasites represent a wide variety of highly adaptive species-specific or wide range infective organisms that temporally or permanently habituate the body surfaces of animals [8,9]. Ectoparasites are vectors or reservoirs that transmit pathogenic agents (bacteria, viruses, protozoa, and helminths) to humans [10,11] and domestic animals [12], some of which present a zoonotic nature [13]. Ectoparasite populations are dependent on host-specific factors, such as population capacity, range, age, sex, behavior, and skin covering [14,15], environmental factors, such as temperature, relative humidity, dew point, geographical location, and seasonal variation [16], as well as the nutritional, developmental, and maturation requirements of the parasite [17].
Rodent ecology and distribution are important factors affecting infectious and zoonotic disease dynamics, risk of transmission, and development in rodents, animals, and humans [21,22]. The distribution and ecology of rodents and rodent ectoparasites that contribute to the high-risk transmission of infectious and zoonotic diseases in North Sinai, Egypt remains mysterious.
The current cross-sectional study investigated the ecological distribution of rodents to identify the species colonizing the study area in North Sinai governorate, Egypt. In addition, we conducted period prevalence (Pp) and risk assessments of ectoparasitic infestations in rodents collected from September 2019 to August 2020, considering seasonal variation.

Ethical approval
The Scientific Research Ethics Committee for the animal, poultry, and lab animal research, Faculty of Veterinary Medicine, Suez Canal University, Egypt, approved the materials, protocol, and study design (approval number 2021014).

Study period and area
The study was carried out from September 2019 to August 2020. The study was conducted in North Sinai, Egypt. The study area was located in the northeast (33.6176° E, 30.2824° N), bound to the north by the Mediterranean Sea with great dimensions up to 220 km (130 miles) from east to west. The study area was 27.574 km 2 , representing approximately 2.7% of Egypt's area ( Figure-1). North Sinai governorate is characterized by a unique Mediterranian climate, consisting of desert and semi-desert regions. Average temperatures and relative humidity vary from 10°C and 18% in the winter and 24°C and 96% in the summer. The northward region receives more rain, which lasts for 3.8 months (November 13-March 7) and accumulates an average of 20.3 mm total precipitation.
During the study period, four seasons of 3 months each were involved in the study. The duration of each season was defined concerning the general and predominating macroclimatic conditions as follows: Fall from September to the end of November, winter from December to the end of February, spring from March to the end of May, and summer season from June to the end of August.

Study design
A cross-sectional retrospective study was designed to investigate the seasonal variation and geographical distribution of rodent species and their ectoparasites.
During the study period, a total of 300 wire traps were purchased in a monthly pattern and thoroughly cleaned using hot water and quaternary ammonium compound. Traps were baited with a variety of fresh foods, including dried fish, tomato, cheese, bread, and slices of cucumber. The trap baits were alternated from time to time to maximize the possibility of catching rodents and overcome the first suspicious impressions from the rodents. Wire traps were distributed in different geographical locations across the North Sinai governorate, such as Baloza, Rabaa, Bir el-`Abd, El-Arish, Sheikh Zuweid, and Rafah (Figure-2). The traps were positioned near residential buildings and poultry, sheep, and goat farms. Traps were distributed before sunset and recollected just before sunrise.

Sampling
A total of 380 rodents were captured during the four successive seasons of the study period. All trapped rodents were enclosed in specially designed plastic bags before euthanasia to ensure the collection of the ectoparasites harbored on their bodies. The rodents were transported to laboratories for identification and external parasite collection.

Rodent laboratory management and identification
In the laboratory, rodents were humanely euthanized using diethyl-ether to perform identification and ectoparasite sampling. Rodents were weighed using a digital scale. The sex and morphological characteristics were recorded, including the head, body, and tail length (mm) for rodent species identification according to Herbreteau et al. [23] and Rahdar and Vazirianzadeh [24]. After the collected rodent species were identified, the frequencies of each species were related to the total number of captured rodents, and seasonal variations during the study period were assessed as recommended by Thrusfield [25].

Ectoparasite collection and identification
Ectoparasites were collected by brushing over the head, neck, trunk, tail, ears, around the eyes, and other parts of each rodent's body using a fine brush. Visible ectoparasites, including those in the transportation bags, were quickly picked up using a toothbrush. Hair particles and ectoparasites were dropped onto a deep white dish or a white sheet of paper. The ectoparasites were counted, grouped by morphology, and fixed in 70% alcohol in sterilized screw-capped clear bottles marked with the date, area of collection, species, and sex of the rodents. Separate bottles were used for each animal host.
The preparation of ectoparasites for light microscopical examination was carried into about Farid et al. [26]. The screw-capped ectoparasite samples were placed into sodium hydroxide 10%, washed using distilled water several times, and dehydrated in serial dilution of alcohol constituting 25%, 50%, 75%, and 100% ethyl alcohol in concentrations. The ectoparasite samples were displaced into Xylene, mounted onto the glass slides with Canada balsam for fleas and lice and Berlese medium for mite, and covered by coverslips. The prepared slides were left for dryness in a hot oven at 40-50°C for 24 h and kept for examination under a light microscope (Barska ® AY13180 Binocular Stereo Microscope, B & H, NY, USA, 10× magnification).
The collected fleas and lice were identified after the available systematic keys of Hendrix [27] and Soulsby [28] and mite after Honey et al. [29] using different keys such as ctenidia, mesopleural rod, and occipital bristle for fleas; dorsal shield and setae for mite, and parategal plate for lice.

Ecological measurement
Ecological macroclimatic conditions, including temperature, relative humidity, and air quality, were recorded regularly in a synchronized pattern with the rodent capture times using digital thermometers (ThermoPro ® TP50 Digital LCD Thermometer Hygrometer Temperature Humidity Meter, ThermoPro, GA, USA) and thermo-hygrometers (Digital Thermometer Hygrometer Indoor Outdoor Temperature Meter Humidity Monitor with LCD Available at www.veterinaryworld.org/Vol.14/November-2021/21.pdf Alarm Clock, 3M Probe Cord, Kanbkam, UAE). The dew points were calculated using the temperature and relative humidity values following Lawrence [30]. The macroclimatic conditions were recorded during the four successive seasons of the study to detect relationships between prevailing weather conditions and parasitic infestations.

Epidemiological measures
We calculated Pp and relative risk (RR) as described by Thrusfield and Christley [31]. We calculated the Pp for the total infestation, fleas, lice, and mites, according to rodent species, rodent sex, and season as follows: Period prevalence (Pp) rate = (α/µ)×100 Where α is the number of (total/specific) infested rodents during a specific period and µ is the number of susceptible rodents in the population (species/sex-specific) during the same period.
RR, that is, the risk of contracting a specific infestation (flea, lice, and mite) during a specific season in the exposed rodent population, was calculated as follows: Where ((a/(a+b)) is the risk that the exposed develop a disease and ((c/(c+d)) is the risk that the non-exposed develop a disease.

Statistical analysis
Statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 software (IBM Corp., NY, USA) [32]. The initial data were analyzed statistically using multifactorial analysis of variance (ANOVA) (two-tailed ANOVA) to determine the influence of seasonal variation, rodent species, and sex on parasitic infestation rates. We used the following statistical model: Where Y ijk is the dependent variable measurement; µ is the overall mean; α i is the fixed effect of seasonal variation, β j is the fixed effect of rodent type and sex, (αβ) ij is the interaction of season and rodent species, and Ɛ ijk is the random error.
Pearson's correlation was conducted to assess the relationships between macroclimatic conditions and rodent distribution and ectoparasitic infestation rates. Correlations were considered strong when r≥0.6, intermediate when 0.6<r≥0.4, and weak when r<0.4. The results were expressed as highly significant when p<0.01, significant when p≤0.05, and non-significant when p>0.05.

Rodent identification and frequencies
In the current study, 380 rodents were captured during the four successive seasons. The numbers were concerning seasons that were; in summer about 186 (106 males and 80 females), fall about 71 (

Parasitological identification
Microscopic examinations revealed three main ectoparasites species: fleas (three identified species), lice (four identified species), and mites (four identified species).
Available at www.veterinaryworld.org/Vol.14/November-2021/21.pdf  The identified flea species were Xenopsylla cheopis ( Figure- (Figure-5c) is characterized by hypostomes with a dorsal labrum of two lobes covered with minute papillae, segmented chelicerae, and pulvillus terminated with two medioventral claws. Ornithonyssus bacoti (Figure-5d) has a dorsal plate that tapers gradually to blunt point setae of the same size and chelicera with no teeth. Table-4 significant (p<0.01) increases in Echidnophaga gallinacean n, X. cheopis, and L. segnis during summer with increased RR during winter. Lice Pp revealed in Table-5 significant   Table-3: Pearson's correlation between ecological macroclimatic conditions with the number of captured rodents in concern with sex (above diagonal) and with the number of infested rodents in concern with sex (below diagonal).   Available at www.veterinaryworld.org/Vol.14/November-2021/21.pdf

Discussion
Rodents are considered a worldwide public health threat as populations increase due to the availability of resources and suitable macroclimatic conditions, including temperature, relative humidity, and dew point in rural and urban areas [33]. Rodent's populations can rapidly grow, resulting in extensive damage to electrical installations, properties, food stores, crops, and grains. The World Health Organization reported that 5-22% of the total global food production is lost by rodent activities [34]. Anthropogenic activities contribute to ecosystem modifications with subsequent changes in the ecological distribution and prevalence of rodents [35]. The parasitism interrelationship between rodents and ectoparasites is complicated. Higher prevalence of rodents might contribute to a higher risk of ectoparasitic infestation resulting in the transmission of more infectious  and zoonotic diseases either directly from rodents or ectoparasites [36,37] or indirectly through exposure to urine, feces, saliva, and blood [38][39][40] or arthropod ectoparasites [41].
In the current study, we collected and identified four rodents: R. norvegicus (brown rat, n=161), R. rattus frugivorus (white-bellied rat, n=119), R. rattus alexandrines (gray-bellied rat, n=50), and M. musculus domesticus (house mouse, n=50). The seasonal prevalence revealed that R. norvegicus, R. rattus frugivorus, and M. musculus domesticus were predominant in the summer, and R. rattus alexandrines was predominant in both the summer and fall. The abundance of rodents in North Sinai is closely related to ecological and climatic conditions, food availability, and socioeconomics of the area. That is why the area suffers from extensive damage to the properties, rodent dropping could be seen everywhere, and some zoonotic diseases with low incidence were recorded. Our findings were consistent with Millán et al. [42] and Pollack et al. [43], who reported that ectoparasites are dependent on rodent survival which in turn usually require moderate temperatures and high relative humidity for survival, multiplication, and development. Yusefi et al. [44] reported that a synchronized pattern recorded a high burden of rodents in some terrestrial areas of Iran for the availability of resources and food. Soliman et al. [45] also noted that environmental conditions, such as the season, topography, and vegetation, as well the availability of food and water resources and hiding places affect rodent hosts and their ectoparasites.
Niche-fulfilling epidemiology has been used to explain the ecological distribution of ectoparasites based on host availability and distribution. Dziemian et al. [46] and Hamidi et al. [47] reported that the moderate atmospheric conditions and food availability that prevail in summer and spring encourage the growth and multiplication of rodents and their ectoparasites that might harbor highly zoonotic agents. Ectoparasitic infestations contribute to anemia and circulatory disorders, secondary infections, irritation, food wastage, lower production and reproduction, skin lesions, hide and wool deterioration that renders them non-marketable, and intoxication in small animals [48]. Moreover, an abundance of emerging diseases is transmitted through ectoparasites [49,50]. Gholipoury et al. [51] recorded the transmission of zoonotic and non-zoonotic parasitic diseases by rodents in Northeastern Iran. Eslami et al. [52] investigated the ectoparasitic infestation prevalence in Rattus rattus of Qeshm Island, Iran, attributing the high rates of infestation to ideal ecological conditions for host growth in the area.
The current study revealed that the Pp of fleas in North Sinai increased during summer in males and fall in females of R. norvegicus, fall in males and summer in females R. rattus frugivorus, and during winter in males and females R. rattus alexandrines and M. musculus domesticus. The identified fleas, E. gallinacean, X. cheopis, and L. segnis, prevailed during summer with an increased RR during winter. Our results were similar to Kowalski and Bogdziewicz [53], who reported that X. cheopis, which transmits Y. pestis and the human plague, contributes to endemic disease in some geographical areas as Southeast Asia. Shahraki et al. [54] also identified X. cheopis in Iran, and Hamidi and Nassirkhani [55] reported similar results and isolated and identified fleas from rodents in Iran. Dahesh et al. [56] reported the risk of rodent fleas in the transmission of Trypanosoma spp. Keskin et al. [57] recovered new host-associated fleas from rodents in Turkey.
The current results also revealed an increased Pp of lice during winter in R. norvegicus, R. rattus frugivorus, R. rattus alexandrines, and M. musculus  [60] recorded four species of chigger mites recovered from rodents in Saudia Arabia. From another perspective, Eladl et al. [61] recorded a high prevalence of mites in laying hen farms in Egypt, reporting residues of pyrethroids in the egg contents.
Rodents can consume a variety of available foods in the ecosystem and tolerate changes well, allowing for rapid population growth [62]. These circumstances make rodents a perfect vector for harboring ectoparasites and transmitting infectious and zoonotic diseases. The extensive activities of humans increase the risk of direct contact with rodents in their habitat, contributing to the transmission of disease [63,64]. Combating rodent infestation should be considered to minimize the risk of transmitting disease through rodents and their infesting ectoparasites [65]. Combating measures can include mechanical control using traditional break-back traps or metal traps and physical barriers [66], biological control using natural enemies without altering the ecological balance [67,68], and chemical control using aluminum phosphide fumigant [69] or anticoagulants [70].

Conclusion
The geographical location and predominating ecological conditions of North Sinai, Egypt, provide a suitable environment that encourages the growth of rodents, including R. norvegicus during summer, R. rattus frugivorus during fall, R. rattus alexandrines during winter, and M. musculus domesticus during spring. Fleas showed different predomination patterns according to season and rodent sex and species. Lice predominated during winter in male and female rodents. Mites predominated during spring in male and female rodents.
Strict preventive and biosecurity measures should be adopted in North Sinai, Egypt, to combat the increased number of rodents and high rates of ectoparasitic infestations. Such measures minimize the risk of transmitting some zoonotic diseases carried by rodents or infesting ectoparasites. Potential control measures might include mechanical, chemical, and biological controls. Overall, the present study established baseline data for rodent species and ectoparasitic fauna in North Sinai, which may facilitate appropriate planning on the control and prevention of rodents and zoonotic diseases in the region.