Prevalence and Seasonal Variation of Trichuris Worms Infection in Mastomys natalensis in Morogoro and Iringa Regions, Tanzania

: Trichuriosis is a disease in mammals caused by the whipworms of the genus Trichuris . These worms are known for the high disease burden they cause in humans and domestic animals, especially in sub-Saharan Africa. In this study, we investigated the prevalence and seasonal variations of Trichuris worms in multimammate rats ( Mastomys natalensis ). The study was conducted between January and November 2021 in Tanzania, in two regions (Morogoro and Iringa) that differ in their eco-climatic conditions. Removal trapping was conducted using Sherman ® live traps during the rainy and dry seasons. The gastrointestinal tracts of captured rodents were screened for the presence of Trichuris worms, which were identiﬁed using morphological keys. A total of 200 M. natalensis rats were collected from each of the regions, with 100 animals in each season. For Morogoro, the overall prevalence of Trichuris worms in M. natalensis was 36% ( n = 72), of which 42% ( n = 42) and 30% ( n = 30) were for the rainy and dry seasons, respectively. For Iringa, the overall prevalence was 65% ( n = 130), of which there were 80% ( n = 80) and 50% ( n = 50) for the rainy and dry seasons, respectively. Trichuris worm infections were signiﬁcantly higher during the rainy season in Iringa than in Morogoro; however, no signiﬁcant difference in infections between males and females was noted in either region or season. Other helminths detected were Strongyloides spp., Capillaria spp., Hymenolepis spp. and eggs of a helminth that has yet to be conﬁrmed, possibly an Anoplocephalid species. Since M. natalensis is the most important pest species in sub-Saharan Africa, and is a carrier of several zoonotic helminths, there is a need for improved surveillance of helminths infections in the studied regions, in order to establish strategic control programs to reduce their adverse impacts on health.


Introduction
Trichuriosis is a disease of mammals caused by nematodes belonging to the genus Trichuris [1][2][3].The disease has spread worldwide, and is most abundant in tropical regions of the world.Infections affect about one billion people around the globe, and more than a quarter of the world's population is predicted to be at risk.In 2020, the World Health Organization (WHO) considered sub-Saharan Africa as one of the regions most heavily affected by Trichuris worms infections.Trichuris worms and other intestinal parasites have received much worldwide attention in recent years due to the exponential increase in their infection rates in humans and animals [4,5].
Approximately 80 species of the worms are currently identified in the genus Trichuris, and most seem to have specific mammalian hosts.For instance, Trichuris muris is a rodent intestinal parasitic nematode that inhabits the large intestine of its host, and induces a strong immune response [5].Indeed, rodents are reservoirs of many zoonotic pathogens, including Trichuris worms [1,6].Mastomys natalensis is among the most abundant and dominant rodent species in sub-Saharan Africa, including Tanzania [7][8][9].This rodent species is highly prolific, and has successfully exploited a wide variety of habitats and environments [7][8][9].In addition, their populations are strongly seasonal, depending on the availability of rainfall and feed abundance [10].
Several studies suggested that differences in environmental conditions, such as temperature and rainfall, lack of access to potable water, poor hygiene and poverty, are major risks for trichuriosis infections [5][6][7][8][9].It is also hypothesized that infections occur more often during the rainy season [11][12][13][14][15]. Consequently, little is known about the prevalence of Trichuris worms in climatically different environments in Tanzania.Therefore, this comparative study aimed to determine the prevalence and seasonality of Trichuris infections in two climatically different regions of Tanzania.The findings will help to determine the influence of season on infection, which can help organize worm control programs, including deworming of vulnerable communities.

Study Sites and Design
A cross sectional study was carried out in two climatically different regions of Tanzania, namely, Morogoro and Iringa, from January to November 2021.In Morogoro, the study was carried out in Choza village close to the Sokoine University of Agriculture main campus, and in Kiroka village (6 • 50 34.9794S; 37 • 38 8.232 E).The villages experience a bimodal rainfall pattern that is characterized by short rains from November to January, and long rains from March to May.The drier season lasts for six months in Morogoro, from May to November [16] (Figure 1).For Iringa, Isimani and Idodi villages were selected (6°50′34.9794′′S; 37°38′8.232′′E), both with a unimodal rainfall pattern.The rainy and dry seasons are well distinguished, with rainfall starting late November or December and ending in April or May, followed by the dry season that starts in May or June and ends in October or November [17].For Iringa, Isimani and Idodi villages were selected (6 • 50 34.9794S; 37 • 38 8.232 E), both with a unimodal rainfall pattern.The rainy and dry seasons are well distinguished, with rainfall starting late November or December and ending in April or May, followed by the dry season that starts in May or June and ends in October or November [17].

Climatic Conditions of Morogoro and Iringa
According to the world climate database, Morogoro has an average temperature of 21.9 • C during the coldest month of July, and 27.3 • C during the warmest month of January; the precipitation amounts to 890 mm per year [16].
In Iringa, the average temperature of the coldest month (July) is 19.0 • C, and that of warmest month (November) is 23.1 • C. The precipitation amounts to 740 mm per year [17,18].

Trapping of Rodents
The removal trapping procedure was conducted in both sites using Sherman ® traps (standard medium size LFA: 7.6 × 8.9 × 23 cm).The trapping was conducted once per month from January to November 2021, during the rainy and dry seasons, for three consecutive nights, making a total of 32 trapping nights.The traps were set in the early evening, and left overnight and inspected the following morning.In each line, the traps were placed 10 m apart from in 10 lines.A total of 300 traps were used in each trapping session, and each trap was baited with peanut butter [19].

Animal Processing and Parasitological Screening for Trichuris Worms
The captured rodents were identified, sexed and weighed before being euthanatized and dissected, in order to obtain the gastrointestinal tract for the recovery of adult worms [20].The intestines and fecal samples collected were preserved in bijou bottles containing 70% ethanol, before being transported to the Institute of Pest Management (IPMC) Laboratory for analysis.
The fecal samples for helminth egg examination were collected from the rectum and recovery of adult worms from the gut contents was carried out after dissecting the entire intestine.The isolation and measurements were conducted using a stereomicroscope mounted with a camera (model MB200, OPTA-TECH, Warsaw, Poland, Oswietienie; 3.3 V) at ×2 magnification [21].
Rodents were considered to be free of Trichuris spp., only after the entire intestinal section was scanned and no eggs or whipworms were seen.The isolated adult Trichuris worms were identified using published morphological keys [22].
Three to four fecal pellets were placed in a 3 mL test tube containing 2 mL of floatation solution (analytical sodium chloride), and once the pellets were softened, they were triturated to break up the pellets to facilitate the release of the helminth eggs.A volume of 1 mL floatation solution was then added to make up a total of 3 mL, followed by covering of the test tube with a coverslip for at least 15 min [23].
The fecal ova were examined at ×100 magnification using a light microscope with a mounted camera (Nikon digital camera DXM1200C).Samples were considered positive for Trichuris spp.when one or more eggs with the characteristic Trichuris "lemon" shape were found [23,24].

Data Analysis
The effects of the geographical region, season, and sex of the rodents on Trichuris worm infections were tested using a generalized linear model with a binomial function.Data entry was carried out using Excel (Microsoft).Data cleaning, statistics and plotting were done in R statistical software 4.1.3[10].

Results
A total of 200 M. natalensis were collected from each region and screened for Trichuris infections.For each season, an equal number of 100 animals were screened.For the Morogoro region, 97 (49%) and 103 (51%) of the animals were males and females respectively.Overall, the prevalence of Trichuris worms infection for both seasons in Morogoro was 36% (n = 72), of which males accounted for 33% (n = 32) while females accounted for 39% (n = 40); moreover, incidences of 22% (n = 22) and 30% (n = 30) were recorded during the rainy and dry seasons, respectively.
For the rainy season, the prevalence were 12% (n = 12) in males and 10% (n = 10) in females, while for the dry season, the prevalence were 16% (n = 16) and 14% (n = 14) for males and females respectively.

Discussion
The current study aimed to determine the seasonal variations of Trichuris worm infections in M. natalensis in two regions of Tanzania (Morogoro and Iringa) that are climatically different.The study showed different infection levels according to the regions and seasons (Table 1, Figure 2).The prevalence of Trichuris worms and other helminths in both

Discussion
The current study aimed to determine the seasonal variations of Trichuris worm infections in M. natalensis in two regions of Tanzania (Morogoro and Iringa) that are climatically different.The study showed different infection levels according to the regions and seasons (Table 1, Figure 2).The prevalence of Trichuris worms and other helminths in both regions was higher during the rainy than the dry seasons.Moreover, the prevalence of Trichuris worm infections was higher in Iringa than in Morogoro.
The relatively high prevalence of Trichuris worms in M. natalensis has also been reported in other studies [1,9], with higher abundances during the rainy season compared to the dry season [7].Our results also suggest that male and female rodents are equally infected.This result is in contrast to other studies that showed females to be more susceptible [27][28][29].This finding calls for more studies to explain this disparity.
During the screening, ova and adult worms belonging to other species were also detected.During the rainy season, the eggs of an unconfirmed helminth, possibly Anoplocephalid species were also found in Morogoro and Iringa, while Strongyloides spp.were detected in Iringa.For the dry season, Strongyloides spp., Capillaria spp., Anoplocephalid species and Hyemenolepis spp.were found in rodents from both regions.This suggests that coinfection with worms of different species is common in rodents.These findings were similar to those of other studies conducted elsewhere, where helminthic infections in mice were screened [28,30].
Furthermore, during this study, the highest prevalence was noted during the rainy season in Iringa.This result agrees with those of previous studies on seasonal variations of Trichuris spp.and other intestinal helminth infections [7,29].However, a study conducted in India by [15] conflicted with this study by showing that Trichuris worm infections were higher during the dry season than the rainy season.
Other studies have shown that Trichuris worm ova can adapt easily under different environmental conditions, thus enabling them to survive well even during dry seasons [29].Various studies have shown that helminth infections in M. natalensis can adversely impair digestive function in a host, and consequently affect its efficiency in absorbing nutrients from the gut.Other reports showed that parasitic infections in M. natalensis can compromise an animal's health, adversely impacting its intestinal microbiota and digestive and immunoregulation [3].
Generally, the examined rodents were more infected with Trichuris worms, Strongyloides spp.and presumptive an Anoplocephalid species with a higher prevalence found in Iringa than in Morogoro.The differences in rainfall pattern, temperature and humidity could cause this variation in favor of the Iringa region.Iringa is located at a higher altitude (1564 masl) compared to Morogoro (254 masl), and has an overall higher rainfall abundance and lower average temperature than Morogoro.These factors may probably be favorable for the survival of the Trichuris ova deposited on the soils in Iringa, and hence a higher chance of infection than in Morogoro.
Based on the findings of this study and other studies [3,31], M. natalensis is shown to be involved in transmitting a series of helminths known to be potential pathogens to humans and animals.Therefore, human, animal and environmental health professionals (One Health) need strategic awareness on control programs to reduce their adverse impact.These programs shall include periodic screening of rodents for helminths, and management of the small mammal populations where there is imminent danger of them causing disease.Moreover, further studies to characterize the different species of Trichuris from different animal species at the molecular level are highly recommended.

Figure 1 .
Figure 1.Map of selected study areas of Morogoro and Iringa regions, Tanzania.

Figure 1 .
Figure 1.Map of selected study areas of Morogoro and Iringa regions, Tanzania.

Figure 2 .
Figure 2. Plot showing the effect of season, location and sex of Mastomys natalensis on Trichuris worm infections in Morogoro and Iringa regions, Tanzania.

Figure 2 .
Figure 2. Plot showing the effect of season, location and sex of Mastomys natalensis on Trichuris worm infections in Morogoro and Iringa regions, Tanzania.

Table 1 .
Prevalences of helminth eggs detected during the screening of Trichuris worms in the fecal samples of Mastomys natalensis in the dry and rainy seasons in Morogoro region, Tanzania.

Table 2 .
Prevalences of helminth eggs detected during the screening of Trichuris worms in the fecal samples of Mastomys natalensis in the dry and rainy seasons in Iringa region, Tanzania.

Season Dry Season inths Detected Total Prevalence (n = 100) Male Female Total Prevalence (n = 100)
* Yet to be confirmed.

Table 1 .
Prevalences of helminth eggs detected during the screening of Trichuris worms in the fecal samples of Mastomys natalensis in the dry and rainy seasons in Morogoro region, Tanzania.

Table 2 .
Prevalences of helminth eggs detected during the screening of Trichuris worms in the fecal samples of Mastomys natalensis in the dry and rainy seasons in Iringa region, Tanzania.