Evaluation of the Molluscicidal Potential of Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany) against Oncomelania quadrasi (Freshwater Snail), a Vector of Human Schistosomiasis: An Introduction to Natural Way of Snail Control to Prevent Schistosomiasis
A Research Proposal
Presented to the Faculty of Graduate Studies
MINDORO STATE COLLEGE OF AGRICULTURE AND TECHNOLOGY
Alcate, Victoria, Oriental Mindoro
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts in Education
Major in Biological Sciences
Andrea Amor G. Ureta
Table of Contents
Chapter I. THE PROBLEM AND ITS BACKGROUND
Nature and Importance of the Study1
Statement of the Hypothesis10
Time and Place of the Study???
Scope and Delimitation of the Study12
Definition of Terms13
Chapter II. REVIEW OF RELATED LITERATURE
Epidemiology and Prevalence18
Pathogenesis and Morbidity23
Control and Management26
Sandoricum koetjape (Santol)39
Swietenia macrophylla (Mahogany)40
Chapter III. MATERIALS AND METHODS
Collection and Preparation of the Plant Sample42
Ethanol Extract Preparation42
Ethanolic Extraction of the S. koetjape and S. macrophylla42
Phytochemical Analysis of S. koetjape and S. macrophylla Extracts43
Test for Alkaloid43
Test for phenols and tannins43
Tests for flavonoids43
Test for saponins44
Tests for carbohydrates/glycosides44
Test for terpenoids44
Collection and Maintenance of Oncomelania quadrasi44
Preparation of Plant Extracts on Snails45
Histological Examination of Internal Anatomy of the Oncomelania quadrasi46
Proper Disposal Procedures46
Chapter ITHE PROBLEM AND ITS BACKGROUNDNature and Importance of the StudyIntestinal parasitic infections (IPIs) have a worldwide distribution and have been identified as one of the most significant causes of illnesses and diseases among the disadvantaged population (Ngui et al., 2011). According to the World Health Organization (2018), an alarming disease, Schistosomiasis, also known as bilharzia, caused by parasitic fluke which affects cattle, domestic animals, as well as humans, is prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. While Hotez et al., (2015) in his article, stated that the disease is included as one of Neglected Tropical Diseases among the Association of Southeast Asian Nations (ASEAN) where almost 200 million people live in extreme poverty. In addition, the International Journal of Infectious Diseases CITATION Els17 l 13321 (Elsevier Ltd, 2017) named Schistosomiasis as the third most devastating tropical disease globally and is a major cause of morbidity and mortality in Africa, South America, the Caribbean, the Middle East, and Asia.
The earliest instance of human schistosomiasis can be traced in Egypt, where retroactive ELISA testing has confirmed evidence of S. haematobium in an Egyptian adolescent mummy more than 5000 years old CITATION Bar12 l 13321 (Barakat, 2012). Although majority of cases are recorded in Southeast Asia, S. japonicum was first discovered in Japan in 1904. In 1905, the first case in China was noted, followed by the first schistosomiasis incident in the Philippines by 1906. And in 1937, the infection was documented and verified in Indonesia. Though schistosomiasis was established in four countries of the region, transmission of the blood fluke has been interrupted in Japan and no new human cases have been discovered since 1978. Therefore at present, S. japonicum only exists in three Asian countries, namely Peoples Republic of China, the Philippines, and Indonesia (Zhou et al., 2010). Currently, schistosomiasis or bilharzia infects over 200 million people worldwide and results in approximately 25 million disability-adjusted life years lost. In the Philippines, it was estimated that five million people lived in schistosomiasis-endemic areas, while over 800 000 individuals with active infection (Olveda et al., 2013).
There are several geographical strains known to infect human intestines such as Schistosoma japonicum, (predominantly in the People’s Republic of China, the Philippines, and Indonesia) Schistosoma mansoni, (mainly in Africa and South America) and Schistosoma mekongi, (Cambodia and Lao People’s Democratic Republic), while Schistosoma haematobium (common in Africa and some countries in the Middle East) communicates the urogenital Schistosomiasis (Zhou et al., 2010). These parasitic worms are carried by freshwater snail vectors, such as Biomphalaria alexandrina, Lymnaea luteola, and Oncomelania quadrasi CITATION Wor18 l 13321 (World Health Organization, 2018).
In the Philippines, (Lam et al., 2018) schistosomiasis remains a public health challenge in endemic focal areas in 28 provinces. It commonly affects the marginalized group specifically, school-aged children, farmers, and freshwater fishermen. Seven provinces in the country was included in the study of Moendeg et al., in 2017 where the disease is classified as endemic in the country, namely Catarman (in Northern Samar), Gonzaga (in Cagayan Province), New Corella (in Davao del Norte), Irosin (in Sorsogon), Talibon (in Bohol), Alang-Alang (in Leyte) and Socorro (in Oriental Mindoro). Aside from the previously mentioned municipality with regards to the Province of Oriental Mindoro, the International Association for Medical Assistance to Travellers also included the areas surrounding Lake Naujan, including the villages of Pola, Victoria and Naujan (IAMAT, n.d.). Whereas Chua et al., in 2017 identified nine provinces known to have Schistosoma japonicum cases. These provinces include Cagayan Valley, Bohol, Negros Occidental, Leyte, Davao, Davao del Sur, Mindoro Oriental, Northern Samar, and Sorsogon. In the study of Leonardo et al., (2012) Oriental Mindoro ranked first in prevalence of schistosomiasis in Luzon. This was attributed to the environmental suitability brought about by heavy rainfalls which frequently cause the Naujan Lake there to overflow, exposing people to potentially contaminated waters.
Human schistosomiasis is characterized acutely by fever, chills, cough, localized dermatitis, and muscle pain. In a report made by Colley et al., in 2014, they explained the chronic mechanism of the disease. The adult schistosome worms colonise human blood vessels for years, successfully evading the immune system while excreting hundreds to thousands of eggs daily, which must either leave the body in excreta or become trapped in nearby tissues. Trapped eggs induce a distinct immune-mediated granulomatous response that causes local and systemic pathological effects ranging from anaemia, growth stunting, impaired cognition, and decreased physical fitness, to organ-specific effects such as severe hepatosplenism, periportal fibrosis with portal hypertension, and urogenital inflammation and scarring. This accords with the information mentioned in the study of Soares Magalhães et al., in 2014 wherein the researchers stated that Schistosoma japonicum infection is a major cause of anaemia, stunted growth, and chronic abdominal organ pathology including portal vein distension, hepato- and splenomegaly and hepatic fibrosis.
Since the first discovery of the infection, several measures, both preventive and curative, are done to counteract schistosomiasis. At least four approaches to controlling infection have been tried at the community level. These are control of snails, public health education, sanitation, and community-based chemotherapy employing Niclosamide. Selective molluscicide treatment in snail-infested bodies of water at main human contact points is the preferred way to approach controlling snail populations (Mwonga et al., 2015). Also in 2015, King et al., in their study, gave emphasis on the snail control by application of chemical molluscicides, either by blanket or focal treatments. It was the most common approach to snail control at Schistosoma transmission sites in Africa and the Americas during the 1950s–1970s. Although copper sulfate and sodium pentachlorophenate were in use in the 1950s, niclosamide became the compound most frequently studied in published reports after 1961 due to its very low toxicity for humans and livestock and its ability to kill snails, their eggs, and cercariae at low concentrations. Niclosamide was also relatively stable to ultraviolet radiation, with persistent lethal effects for up to 24 hours after application. Meanwhile, WHO’s strategy to control schistosomiasis focuses on reducing transmission of the disease through periodic, targeted treatment with praziquantel through large-scale treatment of affected populations, as well as use of molluscicides for control of local transmission (WHO 2017). This was also supported by the study of Colley et al., (2014) wherein preventive public health measures in endemic regions consist of treatment once every 1 or 2 years with the isoquinolinone drug, praziquantel, to suppress morbidity. In some locations, elimination of transmission is now the goal; however, more sensitive diagnostics are needed in both the field and clinics, and integrated environmental and health-care management will be needed to ensure elimination. Meanwhile, Utzinger et al., (2015) recommends that Preventive Chemotherapy and Transmission should be combined with other interventions, such as improved access to clean water, sanitation and hygiene, information, education and communication, and snail control. Interventions should go hand-in-hand with effective surveillance–response mechanisms, readily tailored to social–ecological systems.
However, Al-Zanbagi noted in his study in 2013, in the schistosomiasis control, molluscicides have a record of accomplishment and breakdown. The high cost of imported synthetic compounds, along with increasing concern over the possibility of snail resistance to these compounds and their toxicity in non- target organisms, have given a new force to the revision of plant molluscicides. He suggested that central control of the snail vector of schistosomiasis may depend on the use of plants with molluscicidal properties which may be uncomplicated, economical and suitable technology. Several studies focused on innovating a natural molluscicide, and even impeding the development of cercariae into adult parasites (Michael et al., 2013; Otarigho and Morenikeji, 2013; Rawi, 2011)
Hence, the current study aims to create and evaluate a natural alternative to chemical molluscicide, that is effective, organic, safe, and inexpensive, utilizing the leaves and bark of locally available plants, specifically, the Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany).
ObjectivesThe main objective of the current study is to create and evaluate the molluscicidal potential of Sandoricum koetjape and Swietenia macrophylla ethanolic extracts, using its bark and leaves, against Oncomelania quadrasi (freshwater Snail), a vector of human schistosomiasis.
Essentially, the study sought to realize the following specific objectives:
To determine the phytochemical compounds such as alkaloids, phenols,
tannins, flavonoids, saponins, carbohydrates/glycosides, and terpenoids
1.1 Sandoricum koetjape (Santol leaves);
1.2 Swietenia macrophylla (Mahogany fruit).
2. Determine the evidences of change in the morphology of the internal
anatomy of the Oncomelania quadrasi (Freshwater Snail):
2.1 before the application of the ethanolic plant extracts;
2.2 after the application of the ethanolic plant extracts.
3. Determine the average mortality rate of the Oncomelania quadrasi
(Freshwater Snail) using the different treatments of:
3.1 Sandoricum koetjape (Santol leaves) ethanolic extracts;
3.2 Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
3.3 Combination of Sandoricum koetjape (Santol leaves) and
Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
4. Determine the significant difference in the average mortality rate of the
Oncomelania quadrasi (Freshwater Snail) using the different treatments
4.1 Sandoricum koetjape (Santol) ethanolic extracts;
4.2 Swietenia macrophylla (Mahogany) ethanolic extracts.
4.3 Combination of Sandoricum koetjape (Santol leaves) and
Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
Statement of the HypothesesThere are no phytochemical compounds such as alkaloids, phenols, tannins, flavonoids, saponins, carbohydrates/glycosides, and terpenoids present in:
1.1 Sandoricum koetjape (Santol leaves);
1.2 Swietenia macrophylla (Mahogany fruit).
2. There are no evidences of change in the morphology of the internal anatomy of the Oncomelania quadrasi (Freshwater Snail):
2.1 before the application of the ethanolic plant extracts;
2.2 after the application of the ethanolic plant extracts
3. There are no notable mortality of the Oncomelania quadrasi (Freshwater Snail) using the different treatments of:
3.1 Sandoricum koetjape (Santol leaves) ethanolic extracts;
3.2 Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
3.3 Combination of Sandoricum koetjape (Santol leaves) and Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
4. There is no significant difference in the average mortality rate of the Oncomelania quadrasi (Freshwater Snail) using the different treatments of:
3.1 Sandoricum koetjape (Santol leaves) ethanolic extracts;
3.2 Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
3.3 Combination of Sandoricum koetjape (Santol leaves) and Swietenia macrophylla (Mahogany fruit) ethanolic extracts.
Scope and DelimitationThis study is both experimental as well as descriptive in nature. It is focused in creating and evaluating a natural alternative to chemical molluscicide, that is effective, organic, safe, and inexpensive, utilizing the leaves and bark of locally available plants, specifically, the Sandoricum koetjape (Santol leaves) and Swietenia macrophylla (Mahogany fruit), against the Schistosome vector, Oncomelania quadrasi.
The study to be conducted for 8 weeks, including the week-long actual application of treatments and observation of the 32 set-ups. The snails will be collected from the different barangays surrounding Naujan Lake. The santol leaves and mahogany fruit will be collected from random areas in Bongabong, Oriental Mindoro. The extraction, as well as application of plants extracts will be conducted in the STEM Laboratory of Bansud National High School, Bansud, Oriental Mindoro. While the specimen analysis of morphology of the internal analysis on the Oncomelania quadrasi snail will be sent to Department of Science and Technology.
Definition of TermsTo facilitate a clear understanding of this study, the following terms are operationally defined:
Ethanolic extract. Known as a good solvent for polyphenol extraction and is safe for human consumption (Do, et al., 2014). In the study, two (2) liters of 95% ethanol will be used as extracting solvent where the Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany) barks and leaves will be soaked.
Molluscide. This refers to pesticides which kill mollusks, an animal phylum of tens of thousands of invertebrate creatures (Boyd, 2015). In this study, it is the created through Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany) ethanolic extracts.
Oncomelania quadrasi. An amphibious but mostly aquatic snail host of Schistosoma japonicum in the Philippines CITATION Exp17 l 13321 (WHO, 2017). In this study, the verified species will be exposed to different treatments of Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany) ethanolic extracts.
Phytochemical Analysis. One of the methods that have been used to explore antioxidant compounds in plants (Do, et al., 2014). The test will be done to determine the biologically active compounds present in Sandoricum koetjape (Santol) and Swietenia macrophylla (Mahogany)
Sandoricum koetjape (Santol). A large, ornamental evergreen tree with a dense, narrowly oval crown (Otutu et al., 2016). The verified bark and leaves will be collected, extracted, analyzed, and set in different treatments preparations.
Schistosoma japonicum. A blood fluke resulting to chronic, debilitating disease known as schistosoma (Lam, 2018). In this study, a natural molluscicide will be created to destroy its host snail.
Swietenia macrophylla (Mahogany). An endangered and medicinally important plant indigenous to tropical and subtropical regions of the world (Moghadamtousi et al., 2013). The verified bark and leaves will be collected, extracted, analyzed, and set in different treatments preparations.
Vector. An organism that does not cause disease itself but which spreads infection by conveying pathogens from one host to another (sciencedaily.com). In the current study, the target vector for the disease Schistosomiasis is the Oncomelania quadrasi.
Chapter IIREVIEW OF RELATED LITERATUREThis chapter presents the related principles, framework, and concepts which have direct bearing on the current study.
SchistosomiasisGeographical DistributionIn different countries, different freshwater snails are identified as carriers of the diverse species of the disease. In February 2018, the World Health Organization released the updated information regarding the illness. According to WHO, there are two major forms of Schistosomiasis – intestinal and urogenital. The species responsible for intestinal Schistosomiasis are the following: Schistosoma mansoni – found in Africa, the Middle East, the Caribbean, Brazil, Venezuela, and Suriname; Schistosoma mekongi – several districts of Cambodia and the Lao People’s Democratic Republic; Schistosoma guineensis and related S. intercalatum – rain forest areas of central Africa; and Schistosoma japonicum – China, Indonesia, the Philippines. The urogenital schistosomiasis is caused by Schistosoma haematobium, which are found in Africa, the Middle East, Corsica (France). Each species has a specific range of suitable snail hosts, so their distribution is defined by their host snails’ habitat range. S mansoni and S haematobium need certain species of aquatic freshwater Biomphalaria and Bulinus snails, respectively. S japonicum uses amphibious freshwater Oncomelania spp snails as its intermediate host (Colley et al., 2014). Whereas in a study released by Elsevier in 2017, they identified the countries affected by the said disease and the occurrence in each endemic country. In figure 1, the prevalence in Philippines varies from low to moderate noting that Mindoro is classified within moderate.
Figure 1. Global distribution of schistosomiasis showing the most at-risk populations within Africa. (Elsevier, 2017)
Meanwhile, Yin et al., (2015) conducted a detailed investigation of S. japonicum genetic population structure. Adult individuals of S. japonicum were obtained from twelve different geographical locations, including eight locations across six provinces in mainland China (Anhui, Hunan, Hubei and Jiangxi Provinces in the Yangtze River Basin/lake region, and Sichuan and Yunnan Provinces in the mountainous area of Southwest China) and four other island locations from Asia (Taiwan, Indonesia, Japan and the Philippines). The cercariae were obtained from infected snails, Oncomelania hupensis, and were used to infect rabbits. After 45 days, they removed the adult schistosomes from the mesenteric veins and DNA were extracted and observed. The researchers noted that population from the Philippines, Indonesia and Chinese Taiwan formed distinct genotypes according to their geographical origins. This correlates to another study by Moendeg et al., 2017 which also focused on the geographic strain differentiation in the Philippines utilizing the identified vector Oncomelania hupensis quadrasi right274256500snails in seven endemic municipalities.
Figure 2. Study areas. The map shows the seven endemic municipalities in the Philippines included in this study of Moendeg et al., 2017. They are clustered into three major island groups Luzon, Visayas and Mindanao.
In their study, ten mice were infected percutaneously with 50 cercariae from each municipality. The infected mice were sacrificed six weeks after the infection, and the adult worms were collected from their mesenteric veins and washed with saline for DNA extraction. The gene mapping was done and compared against 10 previously established microsatellite loci by Shrivastava et al., (2003) and Yin et al., (2008) as cited by the researchers. Results showed that though the species was the same, S. japonicum, there was still great genetic diversity in areas with high endemicity.
The study of Yin (2015), as well as Moendeg (2017), suggests that future researches should take into consideration the population diversity/structure differences, as well as possible differences in the response to anti-schistosome vaccines and immunodiagnostics. These findings provide fundamental biological and evolutionary information on S. japonicum, with significant implications for the varying control, intervention, and elimination of this parasite in schistosomiasis-endemic areas. These studies also reveal the diversity of freshwater vectors responsible for the transmission of the trematode as well as the genetic differentiation of the Schistosoma species itself.
Epidemiology and PrevalenceAs mentioned previously, schistosomiasis or bilharzia infects over 200 million people worldwide but in 2014, Colley et al., cited that the estimates made are based on the insensitive egg-detection techniques, which substantially under-represent active infection. Schistosomiasis initiated by infection in early life persists into adulthood, even after infection terminates. Thus, although more than 230 million people are thought to be actively infected with schistosomes, a similar number are in a post-infection stage but continue to have residual morbidity. As a result, the number of people with schistosomiasis (ie, infection related disease) could be closer to 440 million. In Asia, the World Health Organization Regional Office for the Western Pacific (2017) reported that the population at risk is estimated at 80 000 people in Cambodia, where peak transmission season, from February to April, which overlaps with fishing seasons. In China, the reported number of cases in 2016 was 77 194, a 90.23% drop compared to 2014. No acute cases have been reported since 2015. While in the Philippines, 2015 saw another nationwide prevalence survey, which identified one province with over 5% infection prevalence, 12 provinces with a prevalence of between 1% and 5%, and 14 provinces with a prevalence of less than 1%. Although recently, two endemic areas (Gonzaga municipality in Cagayan province, and Calatrava municipality in the province of Negros Occidental), which had not previously been classified as such, were reported through the local health department and confirmed using multiple tools, including the Kato-Katz technique, circumoval precipitation test (COPT), enzyme-linked immunosorbent assay (ELISA), and ultrasound. The emergence of schistosomiasis in areas previously identified as non-endemic has demonstrated the need to improve clinical and laboratory diagnosis and surveillance, along with an effective data reporting system for schistosomiasis cases, with clear clinical case definition to improve clinical diagnosis. Advanced schistosomiasis cases and deaths are now being reported by the media and confirmed by the National Department of Health for Mindanao, Samar, Leyte, and Oriental Mindoro (Olveda et al., 2014).
The Regional Office MIMAROPA in 2016 released information regarding the prevalence in the Region. They identified the four endemic municipalities, all located in Oriental Mindoro, including: Victoria with 13 barangays, Socorro with 8 barangays, Naujan with 7 barangays, and Pola with 5 barangays. They have documented 44,528 individuals belonging to the endemic population.
These studies were also supported by the study of Leonardo et al., in 2012, when the researchers conducted a national baseline prevalence survey of schistosomiasis in the Philippines using stratified two-step systematic cluster sampling design. The objective of the study is to classify the prevalence of schistosomiasis as high, moderate, and low prevalence. Researchers believe that the outcome of the survey will serve as basis for the intervention program to be implemented.
The first two phases of the survey included eight regions in Luzon and the three endemic provinces are included, namely, Sorsogon (Region V), Mindoro Oriental (Region IVB), and Cagayan Valley (Region II) while the nonendemic provinces were randomly selected: Cordillera Administrative Region: Apayao, Region I: Ilocos Sur, Region III: Aurora, Region IV-A: Rizal, and in NCR: 4 cities and 1 municipality. In the second phase, The provinces were used as primary sampling units and the barangays as secondary sampling units. The households were systematically selected and the households represent the clusters within the four phases of the study. The last two phases compared the prevalence between Luzon and Maguindanao.
Figure 3. Prevalence of schistosomiasis in Luzon (Leonardo et al., 2012)
Figure 3 shows the prevalence of schistosomiasis in Luzon with Mindoro Oriental ranking first at 6.3% followed by Sorsogon at 3.6%. Cases were also reported from NCR. These turned out to be migrants from Cagayan Valley.
left22200Figure 4. Age-specific prevalence of schistosomiasis in Luzon (Leonardo et al., 2012)
Figure 4 depicts the prevalence of the disease within specific age groups. shows older age groups with higher prevalence compared to the younger age groups. Peaks of prevalence are noted among the 20–24, 50–59, and 75–79 age groups. Sex-specific prevalence is almost four times higher in males compared to females in Luzon. The researchers therefore concluded that Oriental Mindoro ranks 1 in the prevalence of schistosomiasis in endemic provinces. They attributed this to the high transmission rate brought about by the continuous rainfall and the subsequent floods that facilitate human-parasite contact. Heavy rains frequently cause the Naujan Lake in Mindoro Oriental to overflow exposing people to contaminated waters.
While the map in Figure 5 shows that high prevalence areas are concentrated more in Luzon and the Visayas compared with Mindanao.
1977390000Figure 5. Field data collected by the schistosomiasis research team, College of Public Health, University of the Philippines, Manila (Leonardo et al., 2012)
These studies establishes the varying prevalence of the disease globally and within the country. Most importantly, it denotes the need for intervention in Oriental Mindoro.
Pathogenesis and MorbidityAccording Colley et al., (2014), schistosome eggs, and not adult worms, prompt the morbidity caused by schistosome infections. Many eggs are not excreted and become permanently lodged in the intestines or liver (for S mansoni, S japonicum, and S mekongi) or in the bladder and urogenital system (for S haematobium). The time from initial infection to advanced fibrosis is usually 5–15 years. This was explained further by the studies of Lam et al., (2018) and Olveda et al., (2014), where they cited the three clinical stages in schistosomiasis japonica infection in the Philippines that are recognized and classified: the first or early phase, the systemic or acute phase, and the organ-specific or chronic phase.
The early or first phase begins from the period of cercarial penetration to establishment of paired worms in the mesenteric venules of the intestines. The observed signs and symptoms, though not always manifested for immunized individuals, are localized dermatitis, pruritus, erythema, and a papular rash. The time of onset and intensity of the above-mentioned clinical indicators varies widely.
The systemic or acute phase involves the migration of the larvae through the circulatory system, as well as maturation and pairing of adult worms with the onset of oviposition 6-12 weeks post-exposure and lasts for 2–10 weeks. During this period, infected individuals may have chills, fever, headache, an unproductive cough due to pulmonary involvement, diarrhea or sometimes dysentery, and abdominal cramps. This is attributed to the worm metabolic products that are expelled into the systemic circulation and contribute to a serum sickness-like condition called Katayama syndrome. Moderate to severe disease is characterized by marked eosinophilia, malaise, generalized muscle pain, and pulmonary symptoms and lymphadenopathy. In some cases, Tender hepatomegaly are observed and some splenomegaly are also noted. Neurological symptoms suggestive of meningoencephalitis may be also characterized by the infected person. The severity of clinical disease was also noted to be correlated with the intensity of infection.
The organ-specific chronic phase starts when the adult female schistosomes lay its egg. A variety of clinical manifestations may result from infection depending on the organ involved, and these range from mild to severe, with several gradations in between. In such organs, acute inflammation progressively becomes chronic, and hyperemia and abnormal growths such as polyps and internal hemorrhage are gradually replaced by fibrosis and thickening
of the tissues. Embolization of eggs from the intestine to the liver through the portal system is typical and is responsible for progressive liver fibrosis, portal hypertension, and ascites. Cardiac and renal localizations of lesions are rarely encountered. In the hepatosplenic form, hemodynamic changes are due to S. japonicum eggs trapped in the presinusoidal areas of the liver. These eggs induce classical pipe-stem fibrosis (PSF) around the intrahepatic radicals of the portal vein, leading to increases in splenic pulp and portal vein pressures and signs of severe portal hypertension. The severity of portal hypertension correlates with the severity of fibrosis as demonstrated by Doppler ultrasonography. Patients with severe periportal thickening have dilated portal and splenic veins, high portal vein velocity, and portal vein collateral formation. The chronic pulmonary form is due to eggs that have reached the pulmonary circulation as emboli via the portosystemic
collaterals. The eggs obstruct the arterioles or pass through the walls and lie in the parenchyma just outside the vessels, giving rise to two types of lesions, namely arterial and parenchymatous lesions, and this can lead to pulmonary hypertension or corpulmonale. Bronchial asthma, bronchitis, bronchiectasis, and pulmonary emphysema have also been associated with schistosomiasis. Chronic schistosomiasis of the central nervous system can present clinically with a wide spectrum of signs and symptoms including: headache, nausea, vertigo, visual and speech defects, rigidity, spasm, mental confusion, and hemiplegia. Focal epilepsy due to schistosomiasis in the Philippines has been estimated to be from 2% to 5% among S. japonicum-infected individuals. Ectopic forms of schistosomiasis have been demonstrated in many organs, including the heart, appendix, ovary, fallopian tubes, and uterus.
These studies provides us with information regarding the severity of the disease carried by the snail vector Oncomelania quadrasi. In reducing or possibly eliminating the vectors, the disease infection
Control and ManagementUtzinger et at., (2015) cited the publication of the World Health Organization (WHO) Roadmap (2012) to overcome the impact of neglected tropical diseases in 2020. This includes the reduction of the number of infections in people and elimination of the snails that are required to maintain the parasite’s life cycle.
The researchers cited Bergquist et al. (2009), as well affirming that little attention was paid to the role of diagnostics along with an attempt to highlight different diagnostic tools that can be used for different stages of a schistosomiasis control program. The researchers likewise summarized the different diagnostic procedures for schistosomiasis (Figure 6).
Figure 6. The following grading system was used: x, low; xx, moderate; xxx, high; N/A, not applicable/not available. ELISA, enzyme-linked immunosorbent assay; FECT, formalin–ether concentration technique; IHA, indirect haemagglutination assay; LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction; PHCU, primary healthcare unit (without microscope, centrifuge and other technical equipment); POC-CCA, point-of-care circulating cathodic antigen; UCP-LF CAA, up-converting phosphor-lateral flow circulating anodic antigen (urine-based). (Utzinger et al., 2015)
As seen in Figure 6, several diagnostic procedures are available in determining the severity of the infection. Though there are some disadvantages such as low accuracy of self-report on questionnaires, as well as that only a single pathogen species can be determined and that infections with multiple intestinal parasites cannot be differentiated. The ideal diagnostic approaches such as urine and stool microscopy would allow the concurrent detection of several pathogens in one biological sample.
In the Southeast Asian Report of (2017), they indicated that there is a limited number of diagnostic procedures are available in the region. This includes Kato-Katz thick smear, a conventional stool microscopy, but has poor sensitivity if infection intensities are low especially after anthelminthic treatment. This may leads to low treatment coverage among the true positives.
The participants accordingly agreed that continuation of Mass Drug Administration (MDA), using 40 mg/kg praziquantel, is warranted even in areas that have achieved the criteria for elimination of schistosomiasis as a public health problem until more sensitive and specific rapid diagnostic tools become available for active surveillance of Asian schistosomiasis. Implementation of MDA before the high transmission season, if logistically possible, is considered most effective in reducing transmission. In the Philippines, introduction of praziquantel occurred in 1980, shifting the schistosomiasis control to a chemotherapy-based program. Case finding and treatment led to a decline in the national prevalence of schistosomiasis Olveda et al., 2014).
But Elsevier (2017) called for a need for new global strategy in eliminating Schistosomiasis. The researchers claimed that the annual mass drug administration strategy is failing primarily because the drugs are not getting to the people who need them the most. The current global coverage is 20%, the drug compliance rate is less than 50%, and the drug efficacy is approximately 50% (Figure 7). Thus in reality, only about 5% of the reservoir human population is actually receiving intermittent chemotherapy. Despite claims that more of the drug will soon be made available the current strategy is inherently flawed and will not lead to disease 19455953648elimination.
Figure 7. Percentage of global target population reached with ‘preventive chemotherapy’ for schistosomiasis control. The WHO reported that the global MDA coverage was approximately 20% in 2014. It is assumed the compliance to free treatment is 50% and that the efficacy of PZQ at the single oral dose of 40 mg/kg is 50%.
The researchers recommended an annual treatment of split dose 60 mg/kg PZQ + 6 mg/kg ART. This is due to the rapid reinfection rates are occurring globally and there is now evidence of drug resistance against PQZ and the drugs’ inability to kill migrating schistosomula and the early stages of the disease. The artemether (ART) (and other artemisinin derivatives) additive, which comes from the leaves of the Chinese medicinal plant Artemisia annua, is proven effective against juvenile schistosomes during the first 21 days of infection in both animals and humans, based from the study of Del Villar et al., (2012), as cited by the researcher. Though for advanced cases, the researchers still advise infected individuals to seek appropriate medical and/or surgical follow up.
The recommended 60 mg/kg PZQ + 6 mg/kg ART split dose annual treatment should be implemented along with other activities such as Health Education, improvement of WASH – WAter access (e.g., water quality, water quantity, and distance to water), Sanitation access (e.g., access to improved latrines, latrine maintenance, and faecal sludge management), and Hygiene practices (e.g., handwashing before eating and/or after defecation, water treatment, soap use, wearing shoes, and water storage practices), and Snail control through focal spraying of hotspots. However, the researchers also pointed out that mollusciciding using niclosamide has proven challenging given it is a known pollutant.
Another concern that the researchers identified are that funding and annual evaluation of the control project are the weaknesses of any Mass Drug Administration (MDA) program.
MolluscicideAs previously mentioned, part of Schistosomiasis control is mollusciciding with Niclosamide to control the vector snail that transmits the trematode. Due to the danger brought about by the said synthetic molluscicide, screening of local plants, as well as some strains of bacteria, for molluscicidal activity has received increasing attention.
In 2016, Coelho and Caldeira also pointed out that the use of Niclosamide as molluscicide was recommended by World Health Organization in 1960. However, the researchers pointed out the existence of the molluscs since the Jurassic period and its ability to adapt into its environment. Also, although these snails are monoecious and can self-fertilize, they prefer to reproduce by cross-fertilization when paired. This proliferation and self-fertilization make population control in these snails difficult because one snail can produce 10 million descendants in just 3 months as cited by the researchers. In Brazil, studies to create an alternative molluscicide are continuously pushing through. Various biological and chemical compounds were tested and used as molluscicides. It includes, Bacillus pinotti – a bacteria, but yielded to indefinite result; chemical compounds – copper sulfate, sodium pentachlorophenate, and tritylmorpholine, – proven to be harmful to environment. Niclosamide was identified as the safest and effective choice. A 10% decrease in the prevalence of schistosomiasis was reported in the 1950s. However, commercially available molluscicides have some disadvantages, including not being cost-effective, having lethal effects on other non-target organisms, and requiring frequent application. Efforts are being made to discover molluscicidal products of plant origin, especially potentially biodegradable in nature, due to the growing awareness of environmental pollution. Many plant species, such as Phytolacca dodecandra, Alternanthera sessilis, Jatropha curcas, Euphorbia royleana, E. antisyphilitica, E. lactea ‘Cristata’, E. pulcherrima, E. neutra, Croton tiglium, Codiaeum variegatum, and Solanum xanthocarpum, have been demonstrated to have molluscicidal properties against different snail species. In Brazil, the researchers cited the study of Schall et al. (2008) reported the potential of latex from the plant E. splendens, imported from Madagascar, of being an efficient molluscicidal product, as it is less toxic to other aquatic organisms when compared with niclosamide. Other potential molluscicides are Piplartine – a tropical plant species, which also has less environmental toxicity than niclosamide, and is effective against B. glabrata. The Glinus lotoides has molluscicidal activity against B. pfeifferi snails, and cercariacidal activity against S. mansoni. Although further studies are needed to be conducted to validate the efficacy of the plants.
While in The People’s Republic of China, Li et al., (2016) the snail control program includes environment modification such as burial of surface soil harbouring snails, cement-lining of irrigation ditches, conversion of paddy fields into dry crop farming, utilization of marsh land for vegetables and crops, blocking river branch for aquaculture, reclaiming uncultivated land, weeding for manure collection and so on. They also resorted to tree planting in O. hupensis snail habitats. Chemical mollusciciding, using Niclosamide, is the primary measure in areas where snail habitat modification cannot be implemented temporarily. The researchers also cited that although niclosamide shows good molluscicidal efficacy, its toxicity to fish and other aquatic animals as well as environmental pollution is considerable. Therefore, development of novel molluscicide that is high-effective, low-toxic, cheap and user-friendly chemical molluscicides from plants is always the priority. The plant molluscicides, such as Rongbao and Luowei, show high lethal effect on snails and overcome the disadvantages of niclosamide.
The researchers emphasized the importance of snail control programs to reduce the density of snails in endemic areas and thus significantly lower the risk of schistosomiasis transmission.
In Kenya (Michael et al., 2013), locally available plants such as Azadirachta indica and Entada leptostachya were analyzed for their molluscicidal, cercaricidal and miracicidal activities due to the increasing cases of Schistosomiasis in the area. Plants and roots were collected, dried, and crushed and were extracted using aqueous, methanol and ethyl acetate. 1 kg of the ground powder of each of the extract was extracted with a considerable amount of solvent/distilled water and concentrated in a rotary evaporator while the aqueous extracts were freeze-dried into a thick a gummy extract. Positive control using Niclosamide and negative control using distilled water were set. Phytochemical screening was performed using standard procedures and the various extracts were tested for triterpenes, sterols, flavonoids, saponins, tannins and alkaloids and glycosides.
The collected snails were housed in a temperature controlled snail room (25 – 28ºC) in plastic tanks layered with sterilized sand and gravel. They were fed on dried lettuce and daphnia was used for aeration.
The molluscicidal activity against mature adult and juvenile snails was conducted in accordance with WHO (1965) guidelines. Groups of ten uninfected adult snails were transferred to transparent plastic containers with distilled water and left for 24 hrs. Distilled water was replaced with specified concentrations (1 mg/l, 5 mg/l, 10 mg/l, 15 mg/l, 20 mg/l, 40 mg/l and 80 mg/l) of the 3 extracts from the two plants as well as the controls (1 mg/l of Niclosamide). After exposure to the extracts the snails were given a recovery period of 24 hours in distilled water. Snail mortality was recorded from each set up. Dead snails remained retracted inside their shells at the bottom of the plastic container and failed to show any response to mechanical stimulation with a wooden spatula. Death was confirmed by lack of heart beat. For the miracicidal or larval and cercaricidal activity, larva and the cercariae were exposed to the most active plant extract on juvenile and adult snails. Each preparation was observed under a dissecting microscope for cercariae motility at the following time points: 5, 15, 30 and 60 minutes. Immobile cercariae and miracidia were enumerated and recorded at every time point.
Only methanol extract of E. leptostachya was found to exhibit the highest molluscicidal activity on juveniles and adults with a LD50 (Median Lethal Dose) value of 30.21 mg/l and 40.93 mg/l respectively (P ? 0.05). Methanol extract of A. indica, aqueous and ethyl acetate extracts of A. indica and E. leptostachya were nontoxic to both adult and juvenile snails. On the other hand, methanol extract of E. leptostachya were found to have cercericidal and miracicidal activity. The LT50 (Median Lethal Time) of miracidia and cercariae was 7.69 minutes and 4.25 minutes respectively at a concentration 80 mg/l (P ? 0.05).
Phytochemical screening of the methanol, aqueous extracts and ethyl acetate extracts of A. indica and E. leptostachya confirmed the presence of flavonoids, saponins, tannins, alkaloids, triterpenes and sterols. Results suggest that methanolic root extract of E. leptostachya has molluscicidal activity against Biomphalaria pfeifferi. The results also indicate that methanolic root extract of E. leptostachya have cercaricidal and miracicidal activity against the schistosome larval stages.
Whereas in Nigeria (Otarigho and Morenikeji, 2013), molluscicidal activities of aqueous and ethanolic extracts of leaves of Chromolaena odorata were investigated on adult, one week old juveniles and 3 to 4 days old egg-masses of Biomphalaria pfeifferi. A stock solution was prepared by dissolving 10g of dried powdered C. odorata leaves. The weighed dried powdered parts were soaked in 450 ml of distilled water for 24 hours with occasional vigorous shaking, using magnetic stirrer for the first 6 hours. Then, the suspension was filtered using filter paper. The marc was washed with several portions of distilled water to adjust the volume of the solution using the volumetric flasks to 500 ml. The plant extract was used immediately after the extraction, to ensure their freshness. The same procedure was repeated with 70% ethanol, to obtain the ethanolic extract. After extraction, the solvent was removed by evaporation and the volume adjusted to 500 mL. Ten adults each were exposed to a serial dilution of 40, 80, 160, 240, 320, 400, and 480 ppm distilled water extracts and 20, 40, 80, 160, 240 and 320 ppm ethanolic extracts of leaves of C. odorata. Twenty juveniles of uniform size each were exposed to 8, 20, 28, 40, 80, 120, and 160 ppm of aqueous and ethanolic extracts of the same plant. The adults and juveniles were exposed to the extracts for 24 hours. Thirty eggs each were exposed to 8, 20, 28, 40, 60, 80, and 100 ppm of aqueous and ethanolic extracts for a period of 48 hours. The experiments were repeated twice. LC50 obtained with aqueous extract against eggs, juveniles and adults were 65.75, 75.59 and 217.57 ppm, respectively while these values were 44.03, 44.68 and 88.04 ppm, respectively for ethanolic extract. The LC90 obtained with aqueous extract against eggs, juveniles and adults were 139.54, 249.54 and 288.96 ppm, respectively. These values were 119.03, 123.50 and 245.61 ppm, respectively for ethanolic extract. The plant extracts caused significant (P<0.05) mortality rates in the different stages of B. pfeifferi. Death of the snails was determined and confirmed by the lack of reaction to irritation of the foot with a blunt wooden probe to elicit typical withdrawal movements and absence of heartbeat observed under the microscope, thereafter, mortality counts were recorded. The C. odorata seem to be a promising plant molluscicide candidate and deserve further studies in order to identify and characterize its molluscicidal components.
This study was consistent with the result Rawi’s study back in 2011. Rawi cited in his study that schistosomiasis control has been attempted in several ways including chemotherapy, vector elimination, improved sanitation and health education. However, the successful control of this problem should be based on an integral approach including the control of intermediate host snails. Snail control may be achieved by physical, chemical, and biological methods. Chemical control by molluscicides is performed by using different compounds. However, high costs of chemical molluscicides, the possible built up of snail resistance to molluscicides and their toxicity to non-target organisms has drawn much attention during recent years for the use of plant molluscicides. As reported by several investigators, these represent cheap, safe, locally produced, biodegradable and effective agents in rural areas of developing countries where schistosomiasis is endemic.
In his study, he investigated the effect of some natural plants (Agave filifera whole, Ammi majus flowers and leaves and Canna indica flowers and leaves) as molluscicidal agents and comparing their effects with sulphate salts (CuSO4, ZnSO4, Fe2 (SO4)3 and (NH4) SO4) against Biomphalaria alexandrina snails.
The plant samples were air dried then oven dried at 50°C and powdered by a mixer. They were then extracted using water suspension: wherein for each plant part, weight amounts of the powdered material were added to 1000 mL of dechlorinated tap water to make up the desired of weight/volume concentrations; Cold water: done by soaking ten grams of the powdered plant part in 250 mL of dechlorinated tap water for two days at room temperature, occasionally shaken every 24 hours, and was filtered through filter paper and cold dechlorinated tap water was added to adjust the filtrate at specific volume and replenish any water loss. The adjusted filtrate was used as stock extract to made series of concentrations using appropriate dilutions; Boiled water was also prepared by soaking ten grams of the powdered plant part in about 400 mL of dechlorinated tap water, the suspension then warmed at 100°C for one hour. Boiling tap water was continually added to replenish the evaporated part. The suspension then let to cool at room temperature and filtered through filter paper. The filterate was used as stock to make series of concentrations using appropriate dilutions. The extracts underwent phytochemical analysis to determine the presence of carbohydrates and/or glycosides, cardenolides, saponins, tannins, catchin, flavonoids, and sterols and/or triterpenes.
To test the toxicity of prepared plant extracts, ten snails were immersed in 1000 mL of the experimental concentrations. Three replicates were prepared for each concentration with exposure period for 24 hours. After each period snails were removed, washed thoroughly with dechlorinated tap water and transferred to containers with fresh dechlorinated tap water. Percentage of mortality was calculated against the concentration used. The LC50 and LC90 values of the tested plants were determined as well as the LC10 (sublethal concentration). Histological examination, before and after application of plant extracts, was also done by removing the hermaphrodite gland, fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned. The sections were stained with haematoxyline and eosin. Five replicates were prepared for each test. The exposure period lasts 6 weeks at room temperature (22±2°C). Renewing the experimental concentrations was done weekly to minimize the change in the nature of the tested materials. Microscopical examination of the gonadal tissue of the control snails revealed the presence of male and female gonads, with large nuclei, round nucleolus, surrounded with by follicular membrane, and an outer capsule of fibroplastic connective tissue bound the gonadal tubule.
To determine the snail mortality, the researchers cited the study of Oteifa et al., (1975) that the standard method in which the snails were immersed two or three at a time in 15-20% sodium hydroxide in petridish, if bubbles and blood come out of the shell, it is recorded as alive, if not it is recorded as dead.
Results showed that the water suspension had more potent effect than cold and boiled water extracts of the same plant part. The recorded toxic effects on the longevity of the studied snails mainly attributed to several factors including plant specific differences of the extracted active ingredient, types of extracted products, differences in their mode of action, method of penetration and the behavioral characteristics of the studied animal. Several studies were cited by the researchers to establish that the tested plants’ toxic, as well as its, molluscicidal activity, is due to the biochemical presence of saponin, tannins compounds, triterpenoid, and alkaloid components. Histological examination also revealed destruction in the follicular membrane, degeneration of mature ovum and loss of its nucleolus as well as the nucleus. Tubules were also visibly congested, nucleus appears destructed and irregular, and size of the follicular cavity appears to decrease or is completely disappeared.
All of these studies will guide the current study in determining the important biological active compounds to look for in the plant to be used as well as the different concentrations for the ethanolic extracts.
Sandoricum koetjape (Santol)According to Otutu et al., in 2016, Sandoricum koetjape (Santol or cottonfruit) is an ornamental, evergreen tree, with large tropical fruit. The leaves have sudorific effect and used to make a decoction against diarrhea and fever. The bark is an effective treatment for ringworms, and contains triterpenes with anti-cancer activity; both the seeds and the stems are being studied for the anti-carcinogenic substances they contain. The roots are utilized as anti-diarrheic, anti-spasmodic, carminative, antiseptic, astringent, and stomachicare prescribed as a general tonic after childbirth. The study was aimed to discover the phytochemical constituents and antimicrobial activity of S. koetjape using its leaf and seed extracts. The phytochemical screening revealed presence of some bioactive compounds including saponins, flavonoids, tannins, steroids, and cardiac glycosides. The ethanolic extract of the leaves and seeds showed highest levels of phenols than other secondary metabolites. The extract showed broad spectrum antimicrobial activity. The hot water extracts of leaf and seed produced the highest zones of inhibition against Escherichia coli. The least minimum inhibitory concentration are observed against Staphylococcus aureus and E. coli, Candida albicans, and Streptrococcus pneumonia. These results showed that the leaf and seed extracts exhibited broad spectrum antimicrobial activity.
Swietenia macrophylla (Mahogany)In their study in 2016, Durai et al., described Swietenia macrophylla (Mahogany) as a tropical timber tree. There are numerous reports on the use of S. macrophylla as antibacterial, antifungal, and antiplasmodial agaent. S. macrophylla is also noted as use for treating wound infection and several skin conditions. Presence of limonoids, reported to have antifungal as well as antimalarial properties, was also noted. The leaf, seed, and central fruit-axis was evaluated against gram-positive, gram negative bacteria, and fungi based on the inhibition zone using well diffusion assay. The phytochemical tests exhibited the presence of common phytocompounds such as alkaloids, flavonoids, tannins, terpenoids, glycosides, saponins, volatile oils, amino acids, and proteins as major active constituents. The seed extract had significant level of inhibitory effects on the growth of bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi such as Fusarium sp, Helminthosporium sp and Alternaria sp. The antimicrobial activity exhibited a linear relationship with extract concentrations. The seed extracts proved potential extract against fungal growth.
In the current study, phytochemical analysis will be performed to confirm the presence of the afore-mentioned biologically active compounds.
Chapter IIIRESEARCH METHODOLOGY
This chapter presents the research design, subject of the study, research instrument, scoring and quantification, validity and reliability of the research instrument, data gathering procedure and the statistical treatment of data.
Research DesignFor the current study, an experimental method, complete block design (RCBD) with eight treatments and replication of four times will be employed to determine the molluscicidal activity of Sandoricum koetjape (Santol bark and leaves) and Swietenia macrophylla (Mahogany bark and leaves) ethanolic extracts against Oncomelania quadrasi (Freshwater snail). An experimental design is a problem-solving approach that needs careful manipulation of variables. This is the most prestigious method for advancing Science and Technology because it is production-oriented. While Randomized Complete Block Design uses a group of test plants or animals as subjects which are studied only once but subsequent treatments applied are replicated to determine the cayuse of change (Paler-Calmorin & Calmorin, 2010).
Collection and Preparation of the Plant Sample S. koetjape (Santol bark and leaves) and S. macrophylla (Mahogany bark and leaves) will be randomly collected from Bongabong, Oriental Mindoro. The plants will be sent for identification and authentication at the National Museum-Botany Division, Padre Burgos Ave, Ermita, Manila, Philippines. The bark and leaves of the both S. koetjape and S. macrophylla will be air dried and crushed into powdered form using blender, and will be kept in a dry, clean container separately.
Ethanol Extract PreparationEthanol will be used for extraction because it is known as a good solvent for polyphenol extraction and is safe for human consumption (Do et al., 2013). In another study by Azwanida (2014), ethanolic extract resulted in highest extraction yield with maximum presence of phytoconstituents (alkaloids, saponins, carbohydrates, tannins and flavonoids) compared to the other solvents such as petroleum ether, chloroform and water.
Each forty-four grams (44g) of powdered S. koetjape and S. macrophylla barks and leaves will be soaked and moistened in two (2) liters of 70% ethanol, separately, as an extracting solvent for 48 hours following the method.
Ethanolic Extraction of the S. koetjape and S. macrophyllaThe moistened powdered S. koetjape and S. macrophylla samples will be placed and prepared into the funnels with cover, ready for the maceration set-up. The prepared sample will be then macerated overnight. The extraction will be done using the continuous extraction method using the rotary evaporator apparatus with sufficient amount of 70% ethanol placed. This will be done in Centro Escolar University, Mendiola, Manila.
Phytochemical Analysis of S. koetjape and S. macrophylla ExtractsThe chemical tests for various phytoconstituents present in the extracts will be carried out as described below based on the methods executed by Jaradat, Hussen and Ali (2015).
Test for AlkaloidThe test solution was mixed with little amount of dilute hydrochloric acid and Mayer’s reagent. Formation of a white precipitate indicates the presence of alkaloids.
Test for phenols and tannins
Two milliliter of 2% solution of FeCl3 mixed with crude extract. Black or blue-green color indicated the presence of tannins and phenols.
Tests for flavonoids
Shinoda test: pieces of magnesium ribbon and HCl concentrated were mixed with crude plant extract after few minutes pink colored scarlet appeared that indicated the presence of flavonoids.
Test for saponins
Five milliliter of distilled water was added to crude plant extract in a test tube and it was shaken vigorously. The foam formation indicated the presence of saponins.
Tests for carbohydrates/glycosides
Salkowski’s test: H2SO4 concentrated (about 2 ml) was added to the entire plant crude extract. A reddish brown color produced indicated the entity of steroidal aglycone part of the glycoside.
Test for terpenoids
Two milliliter of chloroform was mixed with the plant extract and evaporated on the water path then boiled with 2 ml of H2SO4 concentrated. A grey color produced indicated the entity of terpenoids.
Collection and Maintenance of Oncomelania quadrasiThree hundred twenty (320) Oncomelania quadrasi will be collected in the area surrounding the Naujan Lake using a flat dip-net scoop. Thirty two (32) set-ups will be made and 10 snails will be used for each set-up. The snails will be housed in a temperature controlled snail room (25 – 28ºC) in plastic tanks layered with soil from the surrounding area of Naujan Lake. They will fed with dried lettuce and gauze will be used for aeration.
Preparation of Plant Extracts on SnailsT1 = 4 mL S.koetjape extract/500 mL tap water; 33%
T2 = 4 mL S. macrophylla extract/500 mL tap water; 66%
T3 = 6 mL S.koetjape extract/500 mL tap water; 100%
T4 = 6 mL S. macrophylla extract/500 mL tap water;
T5 = 8 mL S.koetjape extract/500 mL tap water;
T6 = 8 mL S. macrophylla extract/500 mL tap water;
T7 = 8mL/500mL (4 mL S. macrophylla extract and 4 mL S.koetjape extract)
T8 = negative control tap Water
Eight (8) treatments will be replicated four times in the duration of the study. In each set up, the snails will prevented from crawling out of the troughs by means of a fine mesh white cloth to be used for cover that will be tied to trough by rubber band. The snails will not fed during the course of the experiment; it had been observed that healthy snails live up to five days or more without food (Adetunji and Salawu, 2010) as cited in the study of Otarigho and Morenikeji in 2013, provided other environmental conditions are constant. After 24 hours exposure to the different plant extract concentrations, the snails will be transferred to fresh tap water and maintained there for another 24 hours. Death of the snails will determined.
Histological Examination of Internal Anatomy of the Oncomelania quadrasiNine samples of the internal anatomy of the snail will be observed under the microscope. One sample is non-exposed snail to be observed before the implementation of the study and the eight other samples will be form the experimental snails after the implementation of the study. The comparative morphology of the internal anatomy of the snails could explain the possible damage and lethal effect of the extracts.
Statistical AnalysisThe results of the current study will be expressed as means. To determine the significant difference in the number of dead Oncomelania quadrasi among the treatments, One-Way Analysis of Variance (ANOVA) will be used. Post Hoc (Scheffe) test with a significance threshold of 0.05 will be used for the comparison of the treatment groups.
Proper Disposal Procedures
Proper disposal of waste will be strictly monitored. Dead Oncomelania quadrasi will be crushed buried in the ground. Remaining alive Oncomelania quadrasi will be burned. Other materials will be disposed properly following the standard procedure.
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