Survival of human intestinal parasites in experimentally Infected Laboratory animals in Orlu, South Eastern Nigeria

Survival of human intestinal parasites in experimentally Infected Laboratory animals in Orlu, South Eastern Nigeria Immaculata O. Uduchi 1, Okolie Nnaemeka J. C 2, Ambrose U. Opara 2 and Jacinta C. Elo Ilo 3 1. Department of Medical Microbiology, Imo State University Teaching Hospital, Orlu, Nigeria 2. Department of Medical Laboratory Science, Imo State University, Owerri, Nigeria 3. Department of Paediatrics, Nnamdi Azikiwe University Nnewi Campus, Anambra State, Nigeria Abstract: The survival of human intestinal parasites in experimentally infected Laboratory animals was studied in Orlu, South Eastern Nigeria. A total of 250 male and female patients aged 18-50 years and presenting with gastroenteritis and abdominal discomfort, were selected for the study. Out of this number, 20 declined and 230 participated in the study. They were given questionnaires to complete and specimen containers to produce stool samples. Laboratory examination showed that out of 230 male and female patients, 157 (68.26%) were infected with intestinal parasites. Fifty five (23.90%) were infected with Necator americanus, 32 (13.90%) were infected with Ascaris lumbricoides, 20 (8.69%) were infected with Taenia species, 7 (3.04%) were infected with Trichuris trichiura, 4 (1.74%) were infected with Hymenolepsis nana, 25 (10.87%) were infected with Entamoeba histolytica and 14 (6.09%) were infected with Trichomonas hominis. The most prevalent intestinal parasite in Orlu is Necator americanus (23.90%) while the least prevalent intestinal parasite is Hymenolepsis nana (1.74%). The prevalence of intestinal helminthes 118 (51.3%) was higher than that of intestinal protozoa 39 (17%). Age related prevalence of infection showed that infection decreases as age of patients increased. The highest prevalence of infection 83.78% was amongst patients aged 18 25 years while the least 50% was amongst patients aged 41 to 50 years. Sex related prevalence of infection showed no significant difference (p > 0.05) in the prevalence of intestinal parasite infections between male and female patients. A total of 400 male and female Wistar strain of Rattus albus aged 2-3months and weighing between 80-180g were used for the study. They were purchased from accredited animal house at Emii Veterinary Farm, Owerri, Imo State, Nigeria. The animals were quarantined in a separate compartment shelves netted with barbwire in the animal house of Faculty of Medicine, Imo State University, Orlu Campus. Out of 322 uninfected laboratory animals experimentally infected with different human intestinal parasites (Ascaris lumbricoides, Necator americanus, Trichuris trichuira, Hymenolepsis nana, Entamoeba histolytica and Trichomonas hominis), 34 (10.6%) died. A total of 288 (89.4%) survived. Analysis of their stool samples showed presence of ova, cysts and trophozoites of the inoculated parasites. This study has shown that human intestinal parasites can survive and infect laboratory animals such as Rattus albus, making it possible for such animals to be used for in vivo studies involving human intestinal parasites Introduction: Parasitic diseases remain a major public health problem affecting hundreds of millions of people, particularly in tropical developing countries (Tagboto and Townson, 2001). Human parasitic infections affect more than 30% of the human population across the world. They are caused by endoparasites, including protozoa, nematodes, trematodes and cestodes, (NIH, 2012). Intestinal helminths infections are among the most common infections in humans, especially in tropical and subtropical countries (Yadav and Temjenmongla, 2011). Current estimates South pacific Journal of Pharma and Bio Science 41

suggest that over half of the world population is infected with intestinal helminths, and most of the infected people live in remote rural areas in the developing countries (Horton, 2003). These intestinal diseases cause enormous hazards to the health of people, particularly in children, by contributing to malnutrition, anaemia and retarded growth (Hotez et al., 2007). Human gastrointestinal infections are of frequent occurrence in Nigeria. Parasitic infections and cancer are among the most common types of diseases and disorders in laboratory animals. The most common digestive disorders of laboratory rats are caused by intestinal parasites or bacterial infection (Quesenberry and Boschert, 2011). Infection of rat with tapeworms is relatively uncommon (Quesenberry and Boschert, 2011), and there is usually no sign of infection. However, diarrhoea and weight loss may occur with heavy infestation. Pinworms are common intestinal parasites in rats including laboratory rats. They only require one host and are transmitted through infected feaces. Most infected rats have no signs, but a heavy infection of pinworms can cause diarrhoea due to intestinal inflammation (Quesenberry and Boschert, 2011). Protozoa are normally present in the digestive tract of rats and do not usually cause disease. However, in younger and stressed rats, these protozoa can cause intestinal infections. The infection is transmitted by contaminated feaces, and infected rats have diarrhea, lethargy, rough hair coat, weight loss and in severe cases, heavy bleeding that can lead to death (Rahemo et al., 2012). Finally, providing a balanced diet, proper housing and routine veterinary care will help maintain the rats in the best condition possible. It is important to sanitize and disinfect the cage on a regular basis and also eliminate potential sources of infection. The intestinal parasites can be controlled with appropriate drugs, but cannot always be eliminated. Materials and Methods: Study Area This study was carried out in Orlu, Imo State South-Eastern Nigeria. Imo State shares boundary with Anambra State in the North, Rivers State in the South and West and Abia State in the East. Orlu is one of the three senatorial zones in Imo State comprising of 12 out of 27 Local Government Areas in the State. The State has a population of 3,934,899 based on the estimated census figure of 2006 (National Bureau of Statistics, 2006). Majority of the inhabitants are students, public servants, artisans and housewives. The standard of living is average Test Organisms: The organisms used for this study were intestinal parasites: Necator americanus, Ascaris lumbricoides, Trichuris trichura, Hymenolepis nana, Entamoeba histolytica, Trichomonas hominis and Taenia species isolated from stool samples of patients presenting with symptoms of gastroenteritis and abdominal discomfort at GOPD, Imo State University Teaching Hospital, Orlu. Laboratory Animals: The laboratory animals used for this study were male and female Wistar strain of Rattus albus of 2 to 3 months old and body weights 80 to 180g. South pacific Journal of Pharma and Bio Science 42

Collection and Quarantine of Laboratory Animals: A total of four hundred (400) male and female Wistar strain Rattus albus aged 2-3months and weighing between 80-180g were used for the study. They were purchased from accredited animal house: Emii Veterinary Farm, Owerri, Imo State, Nigeria. The animals were quarantined in a separate compartment shelves netted with barbwire in the animal house of Faculty of Medicine, Imo State University, Orlu Campus. The Rattus albus were allowed to acclimatize to the laboratory conditions for a period of two weeks. They were fed with a commercial pelleted poultry grower s mash- diet, Vital Grower s Feed (Grand Cereals Ltd, Jos, Plateau State, Nigeria) (NAFDAC No: A9-0115). Potable water was also given at intervals. Methods: Ethical Approval: Ethical approval for the study was obtained from the Ethical Committee, Imo State University Teaching Hospital. Selection of Patients: A total of 250 male and female patients aged 18-50 years and presenting with gastroenteritis and abdominal discomfort, were selected from the GOPD, IMSUTH, Orlu, between October, 2014 and March, 2015. The patients were approached on person to person contact basis. The objectives of the study were explained to them and their willingness to participate in the study was sought for using a structured questionnaire. Out of 250 selected patients, 20 declined and 230 accepted to participate in the study. They were given a structured questionnaire to complete and indicate their willingness in writing. They were later given specimen containers for the collection of their stool samples. Isolation of Human Intestinal Parasites: A total of 157 intestinal parasites comprising of 32 ova of Ascaris lumbricoides, 55 ova of Necator americanus, 20 ova of Taenia species, 7 ova of Trichuris trichuira, 4 ova of Hymenolepis nana, 25 cyst of Entamoeba histolytica and 14 trophozoites of Trichomonas hominis were isolated from stool samples collected from the selected patients. The stool samples were analyzed using direct wet mount and stool concentration methods (floatation and sedimentation techniques) as described by Chessbrough, (2002). Also, the Harada-Mori cultural method was used to isolate and differentiate between hookworm species, Necator americanus and Ancylostoma duodenale present in the stool samples (Paraj et al., 1993; Rai et al., 1997; Okolie, 2007; John, 2015) The isolated parasites were washed in physiological saline by centrifugation. The faecal sediments containing the isolated parasites were transferred into screw capped bijou bottles. The bijou bottles were stored in the refrigerator at 8 o C until required for use. Pre- inoculation Management and Examination of Laboratory Animals: Healthy Rattus albus without any sign or symptom of cardiac or renal diseases were selected for the study. Rattus albus with heavy infection of intestinal parasites were not experimentally infected. Female Rattus albus aged above 3 months were screened out to avoid including pregnant ones. Prior to inoculation of the laboratory animals with the isolated human intestinal parasites; All the laboratory animals selected for the study, were fed with the same feed (Vital Grower s Feed) and in equal proportion for 2 weeks before commencement of the experiment and while the study lasted. The faecal samples of all the selected laboratory animals were collected into universal containers and analysed parasitologically for the presence of intestinal parasites using normal saline and weak iodine for wet mount and formal ether concentration method as described by Chessbrough, South pacific Journal of Pharma and Bio Science 43

(2002); Obiajuru and Ozumba, (2009), to certify and classify them into infected or uninfected groups. Also, the modified McMaster egg counting technique was used for quantification of helminth eggs in (eggs/g faeces) from individual faecal samples (MAFF, 1986; Bondarenko et al., 2009). The laboratory animals were separated into infected and Non-infected Laboratory Animals. They were also separated according to gender into male and female groups. The laboratory animals (Rattus albus) were weighed using a weighing scale and their weights were recorded in grams (g). Experimental Design: Out of 400 apparently healthy Laboratory animals used in the study, 78 were infected and 322 were not infected. The uninfected laboratory animals were selected for parasite inoculation and anti parasitic effects of the selected plant extracts. The 322 uninfected animals were divided into 3 groups. The selected Laboratory animals were separated into 6 groups according to age, gender and body weight. Inoculation of Human Intestinal Parasites on Laboratory Animals: Forty eight uninfected laboratory animals were selected from each group for inoculation with human intestinal parasites. Each group of 48 animals was divided into 6 sub groups of 8 laboratory animals and inoculated with 1 human intestinal parasite isolated from selected patients. The laboratory animals were infected by oral inoculation using the cyst, trophozoites, ova or larvae of human intestinal parasites harvested from stool samples of selected patients, using the method of Yadav and Temjenmongla, (2011). The inoculated animals were fed and observed in separate single shelve compartments of laboratory animal cages for a period of 12 weeks. Those that died were replaced from the uninnoculated reserved ones. Post Inoculation management and Examination of the Laboratory Animals for Survival of Human Intestinal Parasites: After the inoculation of the laboratory animals with human intestinal parasites, their faecal samples were collected in labelled universal containers and examined in the Parasitology Unit, IMSUTH, Orlu, for intestinal parasites at 4 weeks interval for 12 weeks. Isolated intestinal parasites were identified using their morphological characteristics and compared with standard Parasitology atlas (Jeffrey and Leach, 1985; Karen, 2005). The parasites were concentrated and stored in appendoff tubes preserved in the refrigerator at 8 0 C for future studies and reference purposes. Results: Prevalence of Intestinal Parasites Amongst Patients in Orlu: Out of 230 male and female patients examined, 157 (68.26%) were infected with intestinal parasites. Specifically, 55(23.90%) were infected with Necator americanus, 32 (13.90%) were infected with Ascaris lumbricoides, 20 (8.69%) were infected with Taenia species, 7 (3.04%) were infected with Trichuris trichiura, 4 (1.74%) were infected with Hymenolepsis nana, 25 (10.87%) were infected with Entamoeba histolytica and 14 (6.09%) were infected with Trichomonas hominis. The most prevalent intestinal parasite in Orlu is Necator americanus (23.90%) while the least prevalent intestinal parasite is Hymenolepsis nana (1.74%). Also, the prevalence of intestinal helminthes 118 (51.3%) was higher than that of intestinal protozoa 39 (17%) (Table 1). Age related prevalence of infection showed that 31 (83.78%) out of 37 patients aged 18 25 years were infected with intestinal parasites. Out of 50 patients aged 26 30 years, 40 (80%) were infected, 46 (73.01) out of 63 patients aged 31 40 years were infected and 40 (50%) out of 80 patients aged 41 to 50 years were infected with intestinal parasites. The prevalence of infection decreases as age of patients increased. The highest prevalence of South pacific Journal of Pharma and Bio Science 44

infection 83.78% was amongst patients aged 18 25 years while the least 50% was amongst patients aged 41 to 50 years as shown in table 2 Sex related prevalence of intestinal parasite infections showed that out of 109 males examined, 74 (67.89%) were infected with intestinal parasites and out of 121 females examined, 83 (68.60%) were infected with intestinal parasites. Statistical analysis of the data using chi square showed no significant difference (p > 0.05) in the prevalence of intestinal parasite infections between male and female patients (Table 3). Comparative Analysis of Diagnostic Methods Used: Fig 1 shows the comparative analysis of the prevalence of the parasites as detected by the two techniques used. The stool concentration techniques detected 55 (23.9%) Necator americanus, 32 (13.9) Ascaris lumbricoides, 20 (8.7%) Taenia species, 7 (3.04%) Trichuris trichiura, 4 (1.7%) Hymenolepsis nana, 25 (10.9%) Entamoeba histolytica and 5 (2.3%) Trichomonas hominis while Direct wet mount technique detected 43 (18.7%) Necator americanus, 26 (11.3) Ascaris lumbricoides, 15 (6.5%) Taenia species, 5 (2.2%) Trichuris trichiura, 3 (1.3%) Hymenolepsis nana, 24 (10.4%) Entamoeba histolytica and 14 (6.1%) Trichomonas hominis. The stool concentration technique detected higher prevalence of infections than the direct wet mount except for Trichomonas hominis where direct wet mount detected higher prevalence (6.1%) than concentration techniques (2.2%). South pacific Journal of Pharma and Bio Science 45

South pacific Journal of Pharma and Bio Science 46

Prevalence of Intestinal Parasites in Laboratory Animals: Four hundred laboratory animals (Albino Rats) were examined, 78 (19.50%) were infected with intestinal parasites and 322 (80.50%) were not infected with intestinal parasites. Of the 78 infected, 27 were infected with 2 different parasites each. Generally, 105 intestinal parasites (Hymenolepsis diminuta, Hymenolepsis nana, Toxocara sp. Ancylostoma sp. Strongyloides sp. and Entamoeba species) were isolated from the laboratory animals. The prevalence of intestinal parasites in laboratory animals as shown in table 4: 36 (9.0%) had Hymenolepsis diminuta, 33 (8.3%) had Ancylostoma species, 12 (3.0%) had Toxocara, 9 (2.3%) had Hymenolepsis nana, 9 (2.3%) had Strongyloides and 6 (1.5%) had Entamoeba species. Out of 201 male laboratory animals examined, 40 (19.9%) were infected with intestinal parasites, 12 of these were infected with 2 different parasites each. Out of 199 female laboratory animals examined, 38 (19.1%) were infected with intestinal parasites and 15 of these were infected with 2 different parasites each. Fig 2 summarizes the comparative analyses of the prevalence of intestinal parasites in laboratory animals between male and female animals. As shown, the prevalence of intestinal parasites was higher (19.9%) in male laboratory animals than females (19.1%), but the prevalence of concommittant infections of (2 different) intestinal parasites was higher (7.5%) in females than males (5.97%). Out of 265 laboratory animals aged 2months, 47 (17.7%) were infected with intestinal parasites and out of 135 laboratory animals aged 3 months, 31 (22.7%) were infected with intestinal parasites. Eleven (4.2%) laboratory animals aged 2 months and 16 (11.9%) laboratory animals aged 3months were infected with 2 different intestinal parasites each. Prevalence of intestinal parasite infections increased as age of the laboratory animals increased. However, analysis of the data using chi square showed no significant difference (p > 0.05) in prevalence of intestinal parasitic infection between different age groups of laboratory animals.. Table 5 shows the parasite egg count in the laboratory animal faeces, 51 (65.4%) out of 78 infected laboratory animals comprising of 26 male animals aged 2months, 21 female animals aged 2months and 4 female animals aged 3months had 1 to 3 parasite eggs per gramme of their stool samples. Nineteen (24.4%) animals comprising of 7 male animals aged 3 months and 12 female animals aged 3 months had 4 6 parasite eggs per gramme of their stool samples while 8 (10.3%) laboratory animals comprising of 5 male animals aged 3 months and 3 female animals aged 3 months had 7 or more parasite eggs per gramme of their stool samples. Analysis of the data using ANOVA showed strong positive correlation (p < 0.05) between parasite egg count and age of laboratory animals. Older laboratory animals aged 3months harboured more parasite eggs than younger laboratory animals aged 2 months. Out of 322 uninfected laboratory animals experimentally infected with different human intestinal parasites (Ascaris lumbricoides, Necator americanus, Trichuris trichuira, Hymenolepsis nana, Entamoeba histolytica and Trichomonas hominis), 34 (10.6%) died. A total of 288 (89.4%) survived (table 6). Examination of their stool samples revealed presence of the respective ova, cysts or trophozoites of the inoculated parasite. South pacific Journal of Pharma and Bio Science 47

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Discussion: Human intestinal parasites (helminthes and protozoa) infection has remained a major public health concern in Nigeria and other developing countries. Prevalence of Intestinal Parasites: The present study showed that the prevalence of human intestinal parasites has remained high (68.26%) in Imo State. This finding agrees with previous reports (Ikpeama et al, 2016), who reported high prevalence of intestinal helminthiasis in Owerri, Imo State. Ikpeama et al. (2016) also reported that delay in disposal of refuse around Owerri municipal metropolis led to increasing prevalence of intestinal helminthiasis in the area. Okolie (2007) reported that prevalence of hookworm infection was statistically higher in Owerri (23.3%) than Port Harcourt (13.3%) (p<0.005) and this was related to seasons of study. Sections of the cities with poor drainage and lower standard of hygiene had higher prevalence rates (11.6%, 8%,) than those zones which improved standard of saination and adequate facilities (3.3%,1.3%). The intestinal parasites isolated from patients in this study (Necator americanus, Ascaris lumbricoides, Taenia species, Trichuris trichiura, Hymenolepsis nana, Entamoeba histolytica and Trichomonas hominis) were similar to those reported in past years in Imo State by previous workers (Obiajuru and Ogbulie, 2003). This observation suggests that intestinal helminthiasis is major health challenge in Imo State and has not received adequate attention. This study observed high prevalence of Necator americanus (23.90%) from human stool samples and 8.3% of Ancylostoma duodenale from naturally infected laboratory animals, using the Harada-Mori cultural method. Necator americanus was not isolated from laboratory animals before they were experimentally infected with human intestinal parasites. Globally, Harada-Mori cultural method is commonly used to differentiate species of hookworms. This is because hookworm eggs might be missed during wet film microscopy due to its fragile nature but Harada-Mori culture method allows growth of hookworm larvae indicating that the patient is infected with hookworm. The findings of this present study agrees with previous studies in Nigeria, (Okolie, 2007) who reported prevalence of Necator americanus (90%)and Ancylostodjma doudenale (10%) in Owerri, Imo State as well as Necator americanus (87.5%) and A. doudenale (25.5%) in Port Harcourt, Rivers State. Udonsi et al., (1980) reported prevalence of 88.61% of hookworm infections were due to Necator americanus and 11.79% due to Ancylostoma doudenale in Owerri, South Eastern, Nigeria. In western Nigeria, Adenusi, (1997) working on distribution of Necator americanus and A. doudenale reported single infection of Necator americanus (72.0%), mixed infection of Nectaor americanus and A. doudenale (23.4%) and single infection of A. South pacific Journal of Pharma and Bio Science 49

doudenale (4.5%). Shahid et al., (2010) reported 6.93% samples positive for hookworms, comprising of A. doudenale (11.50%) and Necator americanus (88.50%). The prevalence of intestinal parasites amongst laboratory animals in the present study was high (19.50%). The parasites (Hymenolepsis diminuta, Hymenolepsis nana, Toxocara sp. Ancylostoma sp. Strongyloides sp. and Entamoeba species) isolated from the laboratory animals were closely related to those isolated from human beings. This suggests close association between the animals and human beings prior to their being procured for breeding and use in the laboratory. This finding underscores the need for tertiary institutions in Nigeria and the ministry of Agriculture to work jointly and establish laboratory animal breeding farms, where students and researchers could acquire these animals from time to time as needs arise. The need for and use of laboratory animals cannot be over emphasized. In situations where students and researchers cannot easily obtain these animals from safe and reliable places, quality study and learning involving laboratory animals cannot be guaranteed. The major essence of the present study is to determine the survival of human intestinal parasites in laboratory animal so that such animals could be used for in vivo studies involving the human intestinal parasites. In Nigeria and many other developing countries, intestinal parasitic infections are common but drugs for treatment are either scarce or expensive. In Nigeria, many people including health workers do not regard intestinal parasite infections as serious health challenge. The need to find and/or develop new affordable drugs is greatly challenged by paucity of research work in this area. These findings of this study suggests that Wister strain of Rattus albus can be used for in vivo studies involving the use of human intestinal parasites. References: 1. Bondarenko, I. G., Kineekova, J., Varady, M., Konigova, A., Kuchta, M. and Konakova, G (2009). Use of modified McMaster method for the diagnosis of intestinal helminths infections and estimating parasitic egg load in human faecal samples in non-endemic areas. Biomedical and life Sciences, Helminthologia, 46 (1): 62-64. 2. Chessbrough, M (2002). District Laboratory Practice in Tropical Countries (Part 1). Cambridge University Press, United Kingdom. P.454. 3. Horton, J (2003). Human gastrointestinal helminth infections: are they now neglected diseases? Trends parasitology,19: 527-531. 4. Hotez, P.J., Molyneux, D.H., Fenwick, A., Kumaresan, J., Sachs, S.E., Sachs, J.D. and Savioli, L (2007).Control of neglected tropical diseases. New England Journal of Medicine, 357:1018-1027. 5. Ikpeama, C.A., Obiajuru, I.O.C. and Ogomaka A.I (2016). The impact of refuse disposal dump sites on the spread of intestinal helminthiasis in Owerri metropolis, Imo State, South Eastern Nigeria. International Journal of Clinical Chemistry and Laboratory Medicine (IJCCLM), 2 (2): 13-18. ISSN: 2455-7153. 6. Jeffrey, H.C. and Leach, R.M (1985) Atlas of Medical Helminthology and Protozoology. 3 rd Edition. Churchill Livingstone, Edinburgh, London/ New York. P. 121. 7. John D.P.G (2015). Incubation of parasitic nematode using Harada-Mori filter paper strip culture method of the fecal sample among children in Teresa St. Stamesa Manila. Public Health Clinical Medicine Diseases and Disorders Health Science Medical Specialties 01. 8. Karen, G.R.N.C (2005). Introduction to the health guide. Rat Health Guide. <http://ratguide.com>. South pacific Journal of Pharma and Bio Science 50

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