Molecular genotypes analysis of Cryptosporidium and Hymenolepis in rats on Lombok Island, Indonesia

Background and Aim: Cryptosporidium parvum and Hymenolepis nana are intestinal parasites that are commonly found in the unclean environment. Their presence in rats promotes the transmission of the cryptosporidiosis and hymenolepiasis to humans or animals nearby. This study aimed to determine the molecular characteristics of C. parvum and H. nana and their distribution in rats on Lombok Island. Materials and Methods: C. parvum and H. nana were investigated in 50 rats from Lombok Island. The molecularparasitological technique used was polymerase chain reaction and sequencing method. Results: From 50 samples of rats’ stool from 10 locations on Lombok Island, 8% (4/50) of C. parvum was detected molecularly with an 18S rRNA gene and 2% (1/50) of H. nana with COX 1 gene. Phylogenetic analysis indicated that C. parvum carrying rats on Lombok Island have a genetic relationship with C. parvum with Obi7 isolates, Japan and H. nana has a genetic relationship with Rodentolepis nana identified with Hn-VT isolates, India. Conclusion: The highest incidence of parasites was found in rats that were caught in the urban areas of Lombok Island, West Nusa Tenggara, Indonesia. Immediately, public health programs in these types of contaminated areas should receive priority attention to prevent further transmission of the parasites from animals to human beings.


Introduction
Diarrhea is a common global disease in both developed and developing countries. The cause of diarrhea generally occurs due to bacterial, fungal, viral, or parasitic infection. Diarrhea if not resolved properly, can cause death and become a significant burden on a country's economic development as well as medical resources. Parasites are one group of pathogens that contribute to diarrhea in developing countries and countries with low economic level. The digestive tract parasites are commonly found in third world countries, especially with tropical climates. The transmission can occur by any kind of animal, and rats are one common carrier [1,2].
Major changes in the natural habitat by humans cause human contact with animals to become more frequent. Diseases related to rats (rodent-borne diseases) began to increase in line with changes in the natural habitat. A rat can act as a transmitter of infectious diseases due to their habitat and habits which are usually scavenging food in dirty places with garbage and consuming water contaminated with diseases so that the rats can cause transmission to not only humans but also animals [3].
Rats can carry various infectious diseases, both groups of viruses, bacteria, and parasites. Infectious diseases could be transmitted to humans through the saliva, urine, and stool or the ectoparasite bites. The dissemination of zoonotic parasites from rats to the human environment requires identification through an early investigation of the zoonotic parasite transmission source. The proximity of the rats with the human environment can be a risk factor for parasite transmission from rats to humans. Parasites can be transmitted through the digestive tract and the infected rats can then cause digestive tract disorders in humans. Parasites of the gastrointestinal tract from rats that have been reported to infect humans include the following, among others: Hymenolepis nana, Giardia lamblia, Balantidium coli, Angiostrongylus cantonensis, Trichuris spp., Enterobius vermicularis, and Cryptosporidium spp. [4][5][6][7].
Seventy-five million people of the world's population are estimated to be carriers of H. nana, and the highest incidence rates can reach up to 25% in some places. Research in Rio de Janeiro, Uttarakhand, North England, and Kuala Lumpur reported that H. nana is commonly found in unclean environments and where are many rats. H. nana commonly infects primary school-age children even with clean living habits [8][9][10][11].
H. nana infection was also found in 35% of the 17 rats in the plantation areas of Lampung with microscopic examination. Molecular identification used ITS-1 gene in 35 adult cestode worms of rats in Lombok and identified two samples of H. nana and one of H. diminuta [12,13].
Cryptosporidium is an intestinal tract protozoan which can be transmitted through the stool of rats. Infections caused by Cryptosporidium can cause moderate to severe diarrhea and even death in children under 2 years old and people who are immunocompromised [14]. Cryptosporidium was found to have a high prevalence of 22.5% (14/62) in immunocompromised children, with microscopic and polymerase chain reaction (PCR) examination [15]. Cryptosporidium was reported in 12.7% (23/180) of Rattus rattus and Rattus norvegicus in Iran using the Ziehl-Neelsen staining [16]. Molecular examination using a small subunit rRNA gene could identify the presence of Cryptosporidium parvum and Cryptosporidium muris in rats in China. C. parvum subtype 11dA15G1 that infects humans was identified after gene sequencing using gp 60 [17,18].
This study aimed to determine the molecular characteristics of C. parvum and H. nana and their distribution in rats on Lombok Island, Indonesia.

Ethical approval and informed consent
This study was approved by the Medical and Health Research Ethics Committee of the Faculty of Medicine, Public Health and Nursing, Gadjah Mada University-Dr. Sardjito General Hospital, with approval number: KE/FK/1222/EC/2018, dated November 21, 2018. Informed consent was obtained from all individual participants included in the study.

Study period, area and sample collection
Fifty samples of rats were caught from five districts in Lombok Island, West Nusa Tenggara Province, from July to November 2019. Sampling was carried out purposively in a tourist-residential area. Each district provided 10 points for each sampling location of rats: Mataram, East Lombok, Central Lombok, West Lombok, and North Lombok. Samples of rat stool were obtained from rat cecum, and DNA of parasites was obtained by extraction used reagents and procedures QIAamp, Fast DNA Stool Mini Kit (Qiagen, Germany).

Parasitological examination technique
DNA extraction, 200 mg of the stool, was mixed with 1 mL InhibitEX Buffer divortex until homogeneous. The lysis process was done by incubating samples in a water bath at a temperature of 85°C for 5 min.
Samples were centrifugated at 10,000 rpm for 1 min to obtain a supernatant. Then, the supernatant as much as 600 µL was added with 25 µL of protein K, then combined with 600 µL of buffer AL and incubated at 70°C for 10 min. To get lysates, 600 µL of ethanol 96-100% was added. After transferring the 600 µL of lysate into the spin column, centrifugated at 10,000 rpm for 1 min, the filtrate was removed and placed in 2 mL tube. A back up of all lysate leftover was made and combined with 500 µL of buffer AW1 into the spin column with a new collection tube, centrifugated at 10,000 rpm for 1 min, the filtrate was removed. Next, 500 µL of buffer AW 2 was added in a new collection tube and centrifugated at 10,000 rpm for 3 min. After removing the spin column from the collection tube, it was installed in a new collection tube and centrifugated at 10,000 rpm for 3 min. Then, it was transferred from the spin column into 1.5 mL tube, then 100-200 µL of buffer ATE was added into the spin column and let stand 1 min to dissolve the DNA and finally centrifugated at 10,000 rpm for 1 min. After discarding the spin column, the 1.5 mL tube which contained DNA was stored at −20°C.
Sequencing, the analysis of the parasite's genetic relationship was done by phylogenetic test, with sample DNA amplification product followed by purification and sequencing. Sequencing used an Applied Biosystems 3500 Genetic Analyzer 2500 with BigDye Terminator kit. Edited DNA parasite sequencing used ClustalW Molecular Evolutionary Genetics Analysis (MEGA) software version 10, USA. The phylogeny tree was based on neighbor-joining [23][24][25][26].  Table-1 and Figure-1.

PCR examination of rats' stool samples on
Available at www.onehealthjournal.org/Vol.6/No.2/5.pdf C. parvum phylogenetic analysis used 18S rRNA gene and H. nana used COX1 to examine and determine the genetic relationship. The analysis of the parasites' genetic relationship used the "Neighbor-Joining" method. Each gene sequence was based on the access available in GenBank. The consensus of the bootstrap tree concluded 1000 replications, and evolution of parasites' distance was calculated based on the method of "Kimura 2." All calculations were performed by phylogenetic analysis using the free software of "MEGA X," USA [23][24][25][26].
C. parvum in rats caught on Lombok Island has a monophyletic kinship with C. parvum (FJ796268.1) in Japan and kinship synapomorphic with C. suis (AB694726.1 and AB449828) in Japan, as shown in Figure- The dissemination of C. parvum and H. nana in urban shown in Figure-4. The dissemination of Cryptosporidium parasite was more than H. nana. C. parvum was found in rats caught in Pemenang Village, North Lombok District, Ampenan and Mataram, Pujut Village in Center Lombok, while H. nana was found only in rats which were caught in Selong, East Lombok District, as shown in Figure-4.

Discussion
C. parvum was most identified in rats which were caught on the Lombok Island, involving 8% of carriers of C. parvum of the 50 rats caught on the Lombok Island. Cryptosporidium is a parasite in animals able to infect humans and can cause diarrhea. One of the worms contained in rats that could infect humans and cause diarrhea is H. nana. Severe infections can cause diarrhea in children and obstruct their development. According to genotyping, from the rats caught on the Lombok Island, there was only one sample with H. nana.
Cryptosporidiosis has been reported in a previous study in the farm areas of the Algerian population. Using microscopic methods with formyl-ether concentration and Ziehl-Neelsen staining, Cryptosporidium was found in moderately high incidence: 36.7% in cattle, sheep 15%, 8.9% of broiler chickens, and 2% in camels. The incidence of cryptosporidiosis was increased to 52.6% in calves with diarrhea [27]. Cryptosporidium was also reportedly found in wild rats caught in the area of Hainan, China. The results of PCR analysis using a small subunit of ribosomal DNA gene found 50% of rats infected by Cryptosporidium from 150 rats which were caught [18]. C. parvum was also reportedly found in two Rattus spp. among 134 rats that were used as laboratory rats, which was detected using PCR of 18S rRNA gene [2]. H. nana is a tapeworm commonly found in rats, and human transmission is caused by unclean living habits, so the embryonated eggs or cysticercoids become swallowed. Several countries have reported the incident of hymenolepiasis, including the

Hymenolepis nana
Mataram  discovery of H. nana in Ethiopia that infected a pregnant women and severe infections with symptoms of abdominal pain, nausea, and diarrhea; the discovery of anemia in Bolivian children infected by parasites and identified as H. nana [28,29]. Gene molecular analysis used COX 1 against rats on Lombok Island, and there was one rat identified with hymenolepiasis out of the 50 rats which were caught. Molecular studies were used in Iraq against R. rattus and found 4% of rats infected with H. nana while in Iran, there was H. nana infection detection of 0.65% in the study population [30,31].
Cryptosporidium and Hymenolepis that were detected on Lombok Island tended to be widely identified in rats caught in urban areas. Parasites generally are found in a congested area with an unclean environment. Cryptosporidium and Hymenolepis which were found in rats can be transmitted to humans or animals nearby. One research report in Bolivia showed a high risk of anemia in children of school age who were infected by H. nana. Continued infection over long periods of time can diminish the performance index and degrade child development [28]. Cryptosporidium infections that cause diarrhea were found in as many as 37% of the 300 samples of diarrhea in Egypt. Patients suspected of cryptosporidiosis should be treated immediately since prolonged infection has been proven fatal in some patients who are immunocompromised. Research recently has shown that the most serious impact is on children or people living with HIV/AIDS [32].

Conclusion
The highest incidence of parasites was found in rats which were caught in the urban areas of Lombok Island, West Nusa Tenggara. Immediately, public health programs in these types of contaminated areas should receive priority attention to prevent further transmission of the parasites.

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