Molecular detection of Trypanosoma lewisi in rodents distributed in dairy cattle pens and residential areas

The World Organisation for Animal Health continues to be concerned about the spread of atypical human-animal trypanosomiasis. Trypanosoma lewisi is one of the rodent-origin zoonotic trypanosomiasis. Despite the fact that many cases of T. lewisi in humans have been documented in several nations, the study of this pathogenic parasite in Indonesia remains limited. The objective of this investigation was to use a molecular approach to detect T. lewisi in rats captured in livestock pens and residential areas. In Pondok Ranggon Village, Cipayung District, East Jakarta, 14 traps were placed in dairy cattle pens and a total of 50 traps were installed outside and inside houses in Lhoksukon and Ceubrek Villages, Lhoksukon District, North Aceh. The captured rats were identified, sedated, and examined for ectoparasites. The blood of rats was then analysed using Giemsa-stained thin blood films and PCR with TRYP1R and TRYP1S primers. The majority of the Rattus species captured in three locations were R. tanezumi (62.07%), followed by R. norvegicus (6.90%) and R. exulans (3.4%). Xenopsylla cheopis were predominant insect found in the present study (60%), followed by Laelaps echidninus (20%), Laelaps nutalli (12%), and Ornithonyssus bacoti (8%). According to the total flea index, rats captured from cattle pens had fewer fleas than rats from other locations. The molecular analysis revealed that T. lewisi was present in 46.15% of rats captured in human settlements and 66.67% of rats captured in the dairy cattle pens. PCR result also demonstrated that positive T. lewisi found higher in inside house (66.67%) compared to outside house (28.57%). It indicates that trypanosome infections are prevalent in rats from domestic environments and cattle pens, and that R. tanezumi can potentially serve as a source of parasites for both animals and humans through fleas acting as vectors.


Introduction
Rats are a synanthropic species that have coexisted with humans since ancient times.They are spread on all cotenintents, except in Antartica.So far, over 1700 species of rodents have been identified to date [1].Rattus tanezumi, R. norvegicus, R. exulans, and other species, are known for being highly adaptable to urban environments.Their distribution becomes more heterogeneous and cosmopolitan as their interaction with humans increases [2,3].
Typically, unhealthy behaviour, poverty, and overpopulation are associated with the emergence of rodent habitat in the surrounding environment, including the traditional livestock management system.Unsanitary conditions in the cages, accumulated dungs, and improper storage of animal feed will result in the formation of rodent nests with the potential to transmit disease, as well as being an ideal environment for the development of disease vectors such as flies, ticks, and fleas.Additionally, residential areas, particularly urban areas, have a high risk of contracting zoonotic diseases.This condition occurs because the area indirectly provides a favourable habitat for certain species of wild animal such as rodents, which will eventually lead to regular and increased contact between rats and humans [4].
Trypanosoma lewisi is Herpetosoma trypanosome known as a cosmopolitan parasite of rats and is primarily spread through fleas.Rattini rodents are the original hosts of T. lewisi, including commensal species, such as the black rat (Rattus rattus), the Pacific rat (R. exulans), and the Norwegian rat (R. norvegicus), including the house rat (R. tanezumi), which have dispersed outside their initial range as a result of human migration and trade [5,6].The parasite is transmitted by several species of rat fleas such as Ceratophyllus fasciatus, Nosopsyllus fasciatus, and Xenopsylla cheopis.The rats were infected primarily through ingestion of infected fleas or contact with faeces of infected fleas [7].The global expansion of T. lewisi has been facilitated by its ability to coexist with rodent hosts.
The T. lewisi infection in humans has been documented and confirmed as a zoonotic disease.Some previous investigations have demonstrated that T. lewisi is capable of transmitting human diseases such as fever, wheezing, edoema, anaemia, and anorexia.The parasite could be classified as a neglected zoonotic pathogen [8] due to the fact that few people are aware of the disease.In addition, a study conducted by Lun et al. [6] on the action of human serum revealed that T. lewisi exhibits resistance to Normal Humans Serum (NHS), confirming its potential as a pathogen in humans.Another study found that T. lewisi also induces an immunosuppressive response in its hosts, hence enhancing their vulnerability to other infections [10].Accordingly, the World Organisation for Animal Health (WOAH) remains to be concerned about the spread of atypical human-animal trypanosomiasis, particularly T. lewisi and T. evansi.
In addition to being a reservoir for the reproduction of T. lewisi, Kocher et al. [11] demonstrated that R. tanezumi is susceptible to T. evansi and capable of carrying the infection over time, based on observations of fluctuating parasitemia and molecular evidence.It has been postulated that R. tanezumi might have a role in the complex epidemiology of surra outbreaks affecting both animals and people.[12,13].
Despite the fact that many cases of T. lewisi have been reported by some contries, the study of this pathogenic parasite in Indonesia remains limited.Lhoksukon and Ceubrek Villages of Lhoksukon District are frequently flooded in North Aceh.This region is notorious for its relatively high rodent population and has a high risk of leptospira infection.Unlike the two villages above, Pondok Ranggon in Cipayung District, East Jakarta is a center dairy cattle farm area in urban region.There are some traditional farms near to residential areas where leptospira cases have been reported.So far, leptospira infection has been the primary focus of research on rodents in these regions.However, the current investigation offers a different perspective.The purpose of this investigation was to determine whether rats in these regions also harbour the blood protozoan pathogen T. lewisi.Based on both parasitological and molecular analysis, there have been no reports of the detection of T. lewisi at these three locations.The present study is the first report of T. lewisi found in Lhoksukon, Ceubrek and Pondok Ranggon villages.

Materials and methods
This study was approved by Ethical Clearance Committee of Faculty of Veterinary Medicine, University of Gadjah Mada, Indonesia with certificate No. 055/EC-FKH/Eks/2023.
The research design employed was accidental sampling, which is a sampling technique based on coincidence or accident and does not provide equal chances for members of the population to be selected as samples or non-probability sampling [14].

Locations for trapping
This study was conducted in Lhoksukon and Ceubrek Villages, Lhoksukon District, North Aceh, Nanggroe Aceh Darussalam Province (NAD Province), and Pondok Ranggon Village, Cipayung District, East Jakarta, DKI Jakarta Province.
Before establishing the traps, a survey of the capturing location was conducted by observing rat droppings and urine.The trap was set if it was determined that rats had travelled through the area.In the Lhoksukon strict, rats were captured by installing 25 traps inside the house (25 traps around the kitchen) and outside the house (25 traps were placed in the back yard or front yard), whereas in Pondok Ranggon, 14 traps were placed in three dairy cattle farms.The traps used in this study were rectangle-shaped, made of iron, and measured 34 cm, 20 cm, and 15 cm in length, width, and height, respectively.Every day for one week, traps were set at 7 p.m. and collected at 7 a.m.The bait used was salted fish.

Blood collection
Before collecting blood through the cardia, ketamine was used to anaesthetize the rodents.In addition, rats were distinguished by their body mass, body length, tail length, ear length, and hind leg length [15].After the rodents were rendered unconscious, blood was collected by cardiac puncture with a dry needle and 5 mL syringe.The blood was deposited in a tube containing EDTA.Blood was swiftly shaken, then stored at -20 o C for molecular analysis [16].

Examination and identification ectoparasites
The ectoparasites were collected by brushing the entire body of the rodents in the opposite direction of their hair.On a 15 x 30 cm plastic tray containing 70% alcohol, ectoparasites that fell from the rat's body were collected.Furthermore, all ectoparasites were transferred into a vial containing alcohol and labelled.Identification of ectoparasites was carried out under a microscope with 100x magnification based on Ristiyanto et al. [17].
The total flea index in house, out house and cattle pens were calculated using the number of rodents and fleas following the formula below [18] :

Examination of blood protozoa
A thin blood film stained with Giemsa (MDT IR®, Indonesia) were used for the examination of blood protozoa.The blood slides were only generated from samples from Pondok Ranggon due to technical limitations in the field.The blood films were created by taking 3 l of blood from an EDTA tube, placing it on one end of a object glass (slide), and then smearing it with a cover glass to obtain a thin film of blood.The smeared slide left to air-dry and a rapid blood stain reagent was utilised for three minutes in methanol, eosin, and Giemsa solutions.The blood smear preparations were then rinsed with running water and examined under a light microscope at 400x magnification [19].

DNA extraction
DNA extraction from whole blood samples was performed using the Genomic DNA Mini Kit (Geneaid, Taiwan) following the guidelines provided by the manufacturer.The DNA extraction results of each isolate were transferred in a 1.5 mL Eppendorf tube and labelled with the corresponding sample ID.All samples were stored at -20 o C for PCR analysis [20].

Data analysis
At both locations, all data is tabulated in Microsoft Excel and descriptively analysed using tables and percentage calculations.

Animal captured
In this study, a total of 29 animals were captured, including 18 animals from residential areas (50 % from inside house and 50 % from outside house) and 11 animals from farms (63.64 % from Farm A, 9.09 % from Farm B, and 27.24 % from Farm C).Based on their taxonomic classification, 72.41% of captured animals belonged to the order of rodents (Rattus spp), while 27.59% were non-rodents (Suncus murinus) (Table 1).The majority of the rattus species captured in this study were R. tanezumi (62.07%), followed by R. norvegicus (6.90%) and R. exulans (3.4%).Based on the location of the trap installation, more R. tanezumi were captured inside the residence (50%) than outside (33.33%).Rattus norvegicus and R. exulans were captured with the same rate at outside house (5.56 %).In the catle pens, R. tanezumi was captured at a rate of 27.27 %, followed by R. norvegicus at a rate of 9.09%.Although S.murinus had a higher percentage (63.64%), it was not a member of the Rodentia order (insectivores group), so it was excluded from the further calculation.
The Asian house rat, scientifically known as Rattus tanezumi (Temminck, 1844) and belonging to the order Rodentia and family Muridae, is a widely distributed rat species that commonly coexists with humans in East and Southeast Asia.[22].Rats are known to inhabit many environments, encompassing both indoor and outdoor habitats.Nevertheless, in instances where food resources become limited in outdoor settings, rats have a tendency to migrate towards residential areas, and in some cases, even infiltrate indoor spaces, driven by their quest for sustenance [23].Consequently, this behaviour can result in detrimental consequences such as property damage [24].Likewise, R. norvegicus and R. exulans were also discovered in human settlements because they have a large distribution area and a broad food tolerance range, allowing them to adapt readily to their environment.
The results of trapping in the dairy cattle enclosures revealed that S. murinus was the most prevalent species (63.64%).The presence of S. murinus in the dairy cattle pens is believed because of role of S. murinus as an insectivore.The conditions of traditional pens were filthy and littered with dungs and waste, making them an ideal habitat for numerous insects that serve as food for S. murinus.Nonetheless, S. murinus was also discovered in residential areas, indicating that this species is not only well-adapted to consuming insects, but also human dietary waste [25].

Ectoparasites
A total of 25 ectoparasites individuals were collected consisting of 23 insects from the captured rats in residential area (7 samples) and 2 insects from the cattle pens (1 samples).Xenopsylla cheopis are predominant insect were found in three areas (60%), followed by Laelaps echidninus (20%), Laelaps nutalli (12%), and Ornithonyssus bacoti (8%) (Figure 1, Table 2).This result was comparable to a study that found the prevalence of ectoparasites L. echidninus and L. nutalli in captured rats in Sulawesi Province to be 11.38% and 6.82%, respectively [26].According to the total flea index, rats captured from cattle pens had fewer fleas than rats from other locations (Table 3).In addition, R. tanezumi and R. exulans captured inside house and outside house were 60% and 32 %, respectively.This finding was in line with Ristiyanto et al. [27] reporting that house rats R. tanezumi and R. exulans captured in the house habitat were more afflicted with ectoparasites than rats captured in the garden habitat.
Xenopsylla cheopis is known as a vector of T. lewisi.Several studies have demonstrated that high levels of X. cheopis infestation coincide with high rates of T. lewisi infection.In addition, Dahesh et al. [28] discovered a significant correlation between the T. lewisi infection and the presence of Laelaps segnis.The results of the study showed that the majority of T. lewisi-infected rats were infested by X. cheopis.Two rats with positive T. lewisi captured in the diary cattle pens also carried X. cheopis.The presence of fleas in domestic environments and cattle pens poses a risk of T lewisi infection in humans and other rats.

Identification of T. lewisi and molecular analysis
Due to technical difficulties on the field, only 13 rats captured in North Aceh have been successfully collected their blood, and no Giemsa-stained blood smears were produced.However, all rats apprehended in Pondok Ranggon have been collected their blood effectively and were used to create blood-stained smears.The results of blood smear stained with Giemsa and PCR analyses were provided in Table 4. Trypanosoma lewisi has distinct morphological characteristics in comparison to other Trypanosoma species, such as T. brucei and T. evansi (Figure 2A).The length of these entities varies between 21 to 36 m, with an average length of approximately 30 m.The breadth of T. lewisi is around 1.5-2.2m.It possesses a large, oval-shaped kinetoplast measuring 0.7-1 m in diameter, which is located in a marginal position, distant from the posterior extremity by a distance of 5-8 m.The nucleus is near to the anterior extremity.The cytoplasm in its mature state exhibits a distinctive C-shaped morphology, characterised by an outer undulating membrane, a lengthy and conspicuous free flagellum, a sizable oval kinetoplast located at a considerable distance from the posterior extremity, and a compact nucleus positioned towards the anterior region [29].Meanwhile, T. brucei (long slender form) exhibit a length ranging from 17-30 m long and 2.8 m wide.The organism exhibits a highly developed undulating membrane characterized by 3 to 5 convolutions.It possesses a freely moving flagellum located at the anterior end, a centrally located nucleus, a pointed posterior end, and a small kinetoplast positioned at the end but not at the terminal point.Almost exclusively, T. evansi appears in the long form of T. brucei [30].
T. lewisi multiplication in rodents has been thoroughly investigated.After a period of rapid multiplication of trypanosomes (approximately 10 days), they stop growing and their numbers stabilize for several weeks, and then the parasites abruptly vanish from the blood, allowing the rat to develop a strong immunity to T. lewisi infection.Another scenario generated by T. lewisi reduces the impact of the immune response known as mimicry.First, the parasites coat their surfaces with a layer of host protein, ablastin, also known as Ig E, which prevents trypanosomes from dividing or proliferating.After several weeks, the host produces Ig M antibodies that recognize this complement on the parasite's surface, activating the complement system and resulting in the rapid lysis of parasites in the circulation.Thus, blood stains reveal low trypanosome densities or fewer trypanosomes [28].Based on the species of the rats, all T. lewisi was detected in R. tanezumi in three distinct locations using both PCR and blood smear.PCR result also revealed positive T. lewisi found higher in inside house (66,67%) compared to outside house (28,57%) (Table 4).The findings of this study indicate a high prevalence of trypanosome infections in rats inhabiting home environments and dairy cattle pens.Consequently, the R. tanezumi species may serve as potential reservoirs for transmitting these parasites to both livestock and humans.Pumhom et al. [5] also found that R. tanezumi and R. exulans, two species known for their synanthropic behaviour, exhibited a strong association with human habitations.This finding suggests that these species may serve as reservoirs for T. lewisi, posing a significant risk for human infection.
The primer utilised in this investigation was TRYP 1, which amplifies 623 bp of DNA target.Normal PCR products consist of a single DNA strand.Nonetheless, non-specific products may be present on the gel with negative rodents (Figure 2B, lanes 4, 7-10, and 12) as well as additional bands in infected/positive samples (Figure 2B, lanes 1, 2, 3, 5, and 11).Desquesnes et al. [31] explained that the unexpected bands shared similarities with DNA from rodents.
Trypanosoma lewisi must devote heightened attention to a number of factors.The parasite is a welltolerated parasite of rats; cyclical transmission by fleas is a more effective mode of transmission than mechanical transmission (T.evansi); and fleas are common and prolific vectors in rats [5].In addition, none of the veterinary drugs have been effective against T. lewisi to date, so the other protocol utilising a higher dose or other drugs should be studied further [30].
Although infection with T. lewis is non-pathogenic in rodents, this parasite can cause significant alterations in the size, cellularity, and histoarchitecture of the respective host's spleen, liver, lymph nodes, and thymus.These pathological alterations are a result of the elimination of this parasite.This condition results in splenomegaly, lymphadenopathy, hepatomegaly to a lesser extent, and immunosuppression.These symptoms represent the clinical manifestations of trypanosoma pathogens in humans and animals [7].
Infection rates of T. lewisi in rats detected in Lhoksukon, Ceubrek and Pondok Ranggon are presented for the first time.Lhoksukon is one of the subdistricts in North Aceh that frequently experiences flooding [33].The majority of research in this region is always connected to its potential as a leptospira transmission source carried by rats.In contrast to Pondok Ranggon, which is known as a dairy cattle centre and is located in an urban area.There have been reports of cases of bloody urine in dairy cattle.So far, rat investigations conducted at both locations have only highlighted their potential as transmission spots for leptospira.
In conjunction with livestock, rodents can act as a reservoir for T. evansi, hence augmenting the potential for infection in both animals and humans.In previous investigations, the existence of T. evansi and T. lewisi in rodents was documented [17,33].This study captures the potential role of rodents in atypical human trypanosomosis caused by animal trypanosomes in relation to their natural habitat.The investigation of rodent infection in various habitats and locations may allow for the evaluation of the specific impact of T. lewisi transmission [5].
According to the findings in the present study, T. lewisi, which causes atypical human trypanosomiasis, has the potential to be transmitted by rats circulating in these areas, both indoors and outdoors, as well as in livestock pens via fleas as the vector.These results are anticipated to provide new insights and early warnings for stakeholders to educate the public about living healthier by maintaining hygiene and sanitising environments.

Conclusion
The prevalence of T. lewisi, which infected rats captured inside the house, is greater than outside the house and in the dairy cattle pens.The presence of X. cheopis fleas carried by rats increases the risk of T. lewisi transmission to humans and other mammals (rodents) in dwellings and cattle enclosures.In order to prevent further spread, it is necessary to conduct regular surveys for rodent and flea control, particularly in public areas.On the level of research, molecular studies should be conducted to identify other vectors that may be responsible for T. lewisi transmission.

Table 1 .
Animals captured by trapping in residential and farms areas in this study.

Table 2 .
Number of ectoparasites identified from the captured rats

Table 3 .
Total flea index of three different locations

Table 4 .
Results of T. lewisi identification based on Giemsa-stained blood smears and PCR analysis