FIRST DATA ON BACTERIAL, FUNGAL AND PARASITIC INFECTIONS OF BLACK RATS ( RATTUS RATTUS ) FROM THE PALM GROVES OF THE ALGERIAN SAHARA

), Microsporum sp. (Pr=19.6%), Trichophyton sp. (Pr=21.7%) and Chrysosporium sp. (Pr=10.9%), noting that saprophytic fungi were the most recorded. On the other hand, according to the richness (S), adults (S = 7) and sub-adults (S = 7) of black rats were the most infested, with leaning for males compared to females, considering all the isolated species as satellites except the Chrysosporium sp. (2.9%) which is presented as a rare species. Concerning parasitic bacteria, aged rats were the most infected followed by adults and sub-adults where total coliforms were present in all individuals of the three classes tested. However, fecal streptococci were noted with a similar infestation rate in all age groups. Unlike this, clostridium sulfite-reducer (CSR) was mostly recorded on aged rats. Concerning the endoparasites found in the intestines of black rats, the pinworms ( Syphacia muris , Syphacia obvelata , and Aspiculuris tetraptera ) were more abundant than the other species. Hence, the current study allowed us to demonstrate that black rats can be considered an important reservoir of several microorganisms that can hold germs and represent a threat to biomedical and veterinary public health.


INTRODUCTION
Rodents are considered among the most frequent and important mammals because they can adapt to different locations and environmental changes (Seifollahi et al., 2016).They act as a vital component in various ecosystems either acting as prey to their predator or as a carrier and reservoir of the diseases (Okoye and Obiezue 2008).It is well recognized that they harbor several ecto and endoparasites thus posing threats to the health of human beings who live close to rodent populations (Namue and Wongsawad, 1997;Zain et al. 2012).For this, in isolated ecosystems, most of these studies have been targeting the parasite helminth fauna and the potential role of rodents as reservoirs of parasitic zoonoses (Casanova et al. 1996, Miquel et al. 1996, Waugh et al. 2006, Milazzo et al. 2010).Knowing that rodents share their food and habitat with humans, secures the transmission of zoonotic pathogens to humans via their urine, feces, hair, and saliva (Meerburg, 2010).Among these dangerous animals, we have chosen to study the associated parasites (intern and extern) of black rats (Rattus rattus), known for sharing their habitat and food with humans.Hence, we have been interested in analyzing the presence of protozoa, helminths, bacteria, and dermatophyte fungi that could be transmitted to humans and cause infectious diseases.The present work is unprecedented in Algeria and provides the first data on the parasite biodiversity in R. rattus.

Study area
This study was conducted in palm groves (Fig 1) and stocks (date hangars located in different palm groves) (Fig 2) from the region of Touggourt (33° 02' to 33° 12' N; 5° 59 'to 6° 14' E) which is located in southeastern Algeria at an altitude of 75 m.It is bordered to the north by palm groves, to the south and east by the Great Eastern Erg, and to the west by dunes of sand.To detect and confirm the presence of parasites (fungi, protozoans, and helminths) associated with black rats, 46 individuals of four age classes were examined (12 adult, 10 old, 12 juvenile, and 12 sub-adult).

Sampling method and identification of rodents
For the trapping of rats, Besancon technology system (BTS) traps were used (eight traps/station) (Mlik, 2019).They were gridded meshes of 26 cm × 12 cm × 14 cm, which were triggered by a hook when the animal touched the bait hooked in the trap.These are very lightweight devices, easy to store and transport.They permit the capture of live individuals which allows very good exploitation of captured animals.Several baits were used including toast, dates, and cheese.Each specimen of R. rattus captured was kept in a numbered jar containing alcohol until manipulation.Once in the laboratory, each individual was examined and identified based on several criteria, notably morphology (coat coloration, soles palmar, and The present study aimed to detect the parasitic fauna associated with black rats (Rattus rattus) from southeastern Algeria.It showed the presence of seven species of parasitic fungi namely Penicillium sp.(Prevalence Pr=91.3%),Aspergillus niger (Pr=91.3%),Alternaria sp.(Pr=58.7%),Cladosporium sp.(Pr=87%), Microsporum sp.(Pr=19.6%),Trichophyton sp.(Pr=21.7%)and Chrysosporium sp.(Pr=10.9%),noting that saprophytic fungi were the most recorded.On the other hand, according to the richness (S), adults (S = 7) and sub-adults (S = 7) of black rats were the most infested, with leaning for males compared to females, considering all the isolated species as satellites except the Chrysosporium sp.(2.9%) which is presented as a rare species.Concerning parasitic bacteria, aged rats were the most infected followed by adults and sub-adults where total coliforms were present in all individuals of the three classes tested.However, fecal streptococci were noted with a similar infestation rate in all age groups.Unlike this, clostridium sulfite-reducer (CSR) was mostly recorded on aged rats.Concerning the endoparasites found in the intestines of black rats, the pinworms (Syphacia muris, Syphacia obvelata, and Aspiculuris tetraptera) were more abundant than the other species.Hence, the current study allowed us to demonstrate that black rats can be considered an important reservoir of several microorganisms that can hold germs and represent a threat to biomedical and veterinary public health.plantar) and craniometry (upper and lower molars).Age and confirmation of species were made through examination of the shape and wear of the molar rows of each individual (Barreau et al., 1991).The farmers capture all the individuals used in the present study within the control framework against these pests.In addition, the current experiment has the approval of the ethical committee of the University of Kasdi-Merbah, Ouargla, Algeria.

External parasites
Hair specimens of four parts of the body (back, belly, tail, and vibrissae) were recovered and preserved aseptically in sterile paper.They were rinsed with alcohol, disinfected with 2% sodium hypochlorite (to eliminate saprophytic fungi), rinsed again with distilled water, and then they were dried near a benzene beak.After drying, they were seeded in Petri dishes containing PDA medium and incubated in an oven at a temperature of 37° c.After obtaining fungal colonies in the Petri dishes, fungi were purified and identified by their macro and microscopic aspects with an identification key of Dufresne (2014).

Internal parasites
All captured individuals were dissected, and their digestive tube was retrieved to examine the internal parasitic fauna (bacteria, nematodes, cestodes, etc.).The contents of these tubes were emptied, using two pincers, in a bottle containing 180 ml of distilled water, then agitated for 20 min to allow the separation of the stomach contents.For fecal coliforms, 5 ml was added in a vial (with a bell) containing 50 ml of bromoCresol purple liquid (BCPL) medium and 1ml in the tubes (with bell) containing 5 ml of BCPL medium (single and double concentration with five replicates for each).After, the gas present in the bells was emptied before incubation at 45° C from 24 to 48 h.For streptococci, the same protocol was adopted but with a Roth medium.The presence of these bacteria is recorded as positive when there is acidification of the medium (change of color from purple to yellow) plus the production of gas reported by the bells.For the clostridium sulfitereducer (CSR), introduce in a sterile tube a volume of 20 ml of the crude solution and put in a water bath at 80° C (10 min), then cool them rapidly under cold water (to eliminate the vegetative forms and to keep only sporulated forms.After that, they were inoculated in a vial, containing liver agar (la) with additives (iron alum and sulfite of sodium), and incubated at 45° C for 48 h.The presence of these microorganisms appeared in the form of colonies surrounded by a black halo.
Coproscopies enriched by flotation with water saturated with salt were carried out for each rodent captured: the feces collected in the terminal part of the colon (2 cm) were mixed with the flotation solution and then filtered.The filtrate was then centrifuged at 3500 rpm for 5 min.Then, a few drops of the flotation solution were added to the centrifuge tube until a meniscus formed on which a cover-slid was placed for 20 min, before being fixed on a microscope slide for observation (Dryden et al., 2005;Ballweber et al., 2014).

Parasitic prevalence (Pr%)
The results of this study were exploited by the parasitic prevalence (Pr%) as well as the mean intensity (MI).According to

Statistical analysis
Statistical analysis was performed using STATISTICA software (v.0.20.) and R software (v.4.2.3).We have used parametric tests for normal results, unlike; the non-normal data were treated with non-parametric tests (e.g.ANOVA test for the normal data and Kruskal-Wallis for the non-normal).

Diversity of black rats-associated parasites
The sampling period of R. rattus, from January to July 2017, allowed us to collect 46 individuals (23 rats for each site).This rodent species was infected with seven species of dermatophytes fungi, three bacterial genera, and six species of helminths (Tab 1).

Parasitic infection of black rats
The variety of parasites in R. rattus allowed us to observe that individuals caught in palm groves were infected with 290 parasites and 285 parasites were found in stock rats (Fig 4).Statistical analysis showed that there was no difference (p=0.5271) between the number of parasites collected in the two locations.
Regarding the sex of the black rats, when sampling parasites, no remarkable difference between the two sexes whereas the number of parasites appearing in infected males (mean = 8.2 ind./rat) was more than in females (mean = 5.4 ind./rat).
On the other hand, the statistical analysis ascertained that there was no significant difference between the two sexes (p=0.7495).On the other hand and according to the age classes, the old rats were more infested with parasites than the others, unlike the juveniles.Statistical data ascertain this with a very highly significant difference (p=0.0008) between the number of parasites collected from all the age categories.These results ascertain that whatever the age or sex of black rats, they will be infected with these parasites either fungi, bacteria, or other parasites.

Internal parasites infection in black rats
The results obtained indicated that S. obvelata comes in first position with 56 individuals, followed by Schistosoma sp.(35 ind.).While A. lumbricoides comes last with 14 individuals (Fig 5).Statistically, there was a significant difference (p=0.0120) between the number of black rats infected by these species.

Figure 5 Internal parasites infection in black rats
Concerning the study stations, the black rats captured in the palm groves were the most attacked by the parasites whose number of Syphacia muris, S. obvelata, Schistosoma sp., and Hymenolepis sp. was higher in individuals caught in palm groves than stocks (Fig 6).On the other hand, the number of individuals of Aspicularis tetraptera and Ascaris lumbricoides identified in the black rats from stocks was higher than those collected from palm groves.Statistical analysis showed that there was a highly significant difference (p=0.0029) between individuals from the two sampling stations.

Figure 6 Internal parasite infection in black rats depending on stations
While depending on the sex of R. rattus, the results obtained showed that males were more infected than females.Hence, the continuous movement of male rats over females may explain it.Statistical data showed that there was a highly significant difference between the two sexes (Fig 7).

Dermatophyte fungi infection in black rats
The present experiment, with the isolation of different species of fungi from different parts of black rats' bodies, allowed us to observe the dominance of saprophytic fungi in the four parts.This type of fungi was highly recorded in the tails of these animals, unlike the dermatophytic fungi that were mostly isolated from the belly of black rats tested (Fig 9).Statistical data revealed that there was a very high significant difference between the isolated fungi (p=0.0010).Depending on the body parts examined, the belly was the most part carrying these fungi (31.7%) followed by the tail (28%).Depending on the statistics, there was no difference recorded between these parts (p=0.4232).

Bacterial infection in black rats
The current study showed that the coliforms were more detected in the black rats examined whereas the prevalence of these bacteria was 73.9%, followed by streptococcus and CSR with 41.3% (Fig 10 ).The presence of infected black rats around animal facilities poses high risks not only for animals but also for farmers and their families.Hence, the possibility of these rats contaminating the environment, food, and water source with their parasites poses a public health threat since these rats live in close association with humans.Hence, these parasite infections in the urban rats in this study poses a health risk to human.
The current study allowed us to detect considerable differences in parasites, especially in helminth species.In consequence, black rats could be considered an important vector of several microorganisms that can hold germs and represent a threat to biomedical and veterinary public health.Based on these findings, it is necessary to perform а control way aimed at full rat eradication to prevent zooanthroponosis.

Figure 1 Figure 2
Figure 1 Palm grove stations

Figure 7 Figure 8
Figure 7Internal parasite infection in black rats depending on sex

Figure 10
Figure 10 Prevalence of bacterial infection in black ratsDISCUSSIONAmong the main vectors of disease contamination, not only humans, but also domestic animals (dogs, cats), rodents (rats and mice), reptiles (lizards, margouillat), and insects (flies) can constitute reservoirs for various germs (staphylococci, streptococci, salmonella)(Gwenzi et al., 2021).Rattus rattus is a known carrier of bacteria, viruses, and parasites of zoonotic and veterinary importance(Meerburg et al., 2009;Reperant et al., 2009).In addition, rats transmit diseases directly or indirectly where they are incriminated for deaths more than any other causes (Mohammed Ayyal et al., 2019).Threat to human health is well recognized when potentially life-threatening diseases currently have no specific treatment, cure, or vaccine (Desvars-Larrive et al., 2017).On the other hand, rats are capable of carrying and shedding Escherichia coli (Burriel et al, 2008; Guenther et al., 2010; Nkogwe et al., 2011).Rattus rattus is a serious pest in urban and rural environments.It is the cause of extensive economic damage to crops, stored food, farms, industries and households(Pimentel et al., 2005).Black rat populations also harbor and spread zoonotic pathogens, such as viruses (e.g., Seoul hantavirus), bacteria (e.g., Leptospira interrogans), protozoa (e.g., Toxoplasma gondii) and helminths (e.g., Hymenolepis spp.)(Himsworth et al., 2013).The diversity of internal parasites (bacteria and helminths) could be probably due to the diversity of the arthropods-vector-disease community collected from the same individuals of black rats from southeastern Algeria (same region of study)(Mlik et al., 2022).The same authors have isolated several lice and acari species that were identified on this species are among the main disease vectors.Concerning our finding on helminths, River (2015) has confirmed that the three pinworms found in rats and mice are Syphacia muris, S. obvelata and Aspicularis tetraptera.In addition, Panti-May et al. (2017) found that Hymenolepis diminuta was the most prevalent pathogen, particularly in black rats (14.2%).Meshkekar et al. (2014) declared that R. rattus was the predominant rodent species infected with five different parasites, in Iran, two of which are zoonotic (H.diminuta and H. nana).Furthermore, Mahmood Amin (2019) noted that 43.63% of black rats

Valtonen et al. (1997), the
prevalence (Pr%) is the number of individuals infected with a particular parasite species (np)/number of hosts examined (n).Mean intensity (MI) is the total number of individuals of a particular parasite species in a sample of a host species/number of infected individuals of the host species in the sample (Margolis et

Table 1
Different types of parasites found in black rats from the region of Touggourt Trichophyton sp., Microsporum sp. and Chrysosporium sp., respectively (Tab 1).Concerning the three bacterial genera examined, coliforms (34 infected rats) were most frequently detected in the feces of R. rattus, followed by Streptococcus and CSR with the same number (19 infected rats).Although, the internal parasites, S. obvelata (63.04%) was the most frequently detected species, followed by the Schistosoma sp.(45.65%), while A. tetraptera (19.57%) comes last (Tab 1).Concerning the recorded mean intensity, Alternaria sp., and Cladosporium sp. have presented the most important mean intensities according to the other species, unlike Penicillium sp. which showed the lowest intensity for fungi.On the other hand, A. lumbricoides and Schistosoma sp. have recorded the highest values for nematodes (Tab 1).
(Franssen et al., 2016)al., 2012;Chero et al., 2016)uman hymenolepiasis is a zoonosis caused by the cestodes H. nana and H. diminuta(Nkouawa et al., 2016).It is often asymptomatic but can cause chronic diarrhea, abdominal pain, irritability and itching(Martínez-Barbabosa et al., 2012;Chero et al., 2016).On the other hand, Franssen et al. (2016) declared that S. muris gravid females actively leave their host's intestine and deposit the eggs around their host's anus, after which (auto) infection and transmission between rats take place through grooming and social behavior.Hymenolepis spp.are frequently transmitted to humans and reported as zoonotically important (Khan et al., 2021), whereas H. diminuta is transmitted by ingestion of Tribolium confusum (flour beetle, intermediate host) with contaminated cereals, or by the fecal-oral route.Hymenolepis nana is transmitted through fecal-oral contact (eggs), or by accidental ingestion of intermediate hosts harboring cysticercoids(Franssen et al., 2016).The infections of these species in humans are mostly asymptomatic, although weakness, headache, abdominal pain, and diarrhea may occur in artificial digestion of the diaphragm and both hind legs (CDC, 2016).