Article : More Know about Ascaris lumbricoides

HOSPES & DISEASE NAME

Ascaris lumbricoides, common saying 'soil transmitted helmint' is the largest of the intestinal nematodes parasitizing humans. It has worldwide in distribution and most prevalent through out the tropics,subtropic,and more prevalent in the countryside than in the city.The disease's name is ascariasis.

¨MORFOLOGY

Adult: The adults are cylindrical in shape, creamy-white or pinkish in color. The female averages 20-35cm in length, the largest 49cm. The male is smaller, averaging 15-31cm in length and distinctly more slender than the female. The typical curled tail with a pair sickle like copulatory spines. On the tip of the head there are three lips, arranged as "a Chinese word “ 品They have a complete digestive tract. Reproductive organs are tubular. male has a single reproductive tubule. The female has two reproductive tubules and the vulva is ventrally located at the posterior part of the anterior 1/3 of the body. The three lips are seen at the anterior end. The margin of each lip is lined with minute teeth which are not visible at this magnification.

Egg : There are three type of the eggs. They are fertilized eggs, unfertilized eggs and decorticated eggs. We usually describe an egg in 5 aspects: size, color, shape, shell and content.

Fertilized eggs: broad oval in shape, brown in color, an average size 60× 45µm. The shell is thicker and consists of ascaroside, chitinous layer, fertilizing membrane and mammillated albuminous coat stained brown by bile. The content is a fertilized ovum. There is a new-moon(crescent) shaped clear space at the each end inside the shell.

Unfertilized egg: Longer and slender than a fertilized egg. The chitinous layer and albuminous coat are thinner than those of the fertilized eggs without ascaroside and fertilizing membrane. The content is made of many refractable granules various in size.

Decorticated eggs: Both fertilized and unfertilized eggs sometimes may lack their outer albuminous coats and are colorless.


LIFE CYCLE

This worm lives in the lumen of small intestine, feeding on the intestinal contents, where the fertilized female lays eggs. An adult female can produce approximately 240,000 eggs per day, which are passed in feces. When passed, the eggs are unsegmented and require outside development of about three weeks until a motile embryo is formed within the egg.

After the ingestion of embryonated eggs in contaminated food or drink or from contaminated fingers, host digestive juices acts on the egg shell and liberate the larva into the small intestine. These larvae penetrate the intestinal mucosa and enter lymphatics and mesenteric vessels. They are carried by circulation to the liver, right heart and finally to the lungs where they penetrate the capillaries into the alveoli in which they molt twice and stay for 10-14days and then they are carried, or migrate, up the bronchioles, bronchi, and trachea to the epiglottis. When swallowed, the larvae pass down into the small intestine where they develop into adults. The time from the ingestion of embryonated eggs to oviposition by the females is about 60-75 days. The adult worms live for about one year. The ascarid life cycle is as the following diagram.


Pathogenitas

There are two phase in ascariasis:

1. The blood-lung migration phase of the larvae: During the migration through the lungs, the larvae may cause a pneumonia. The symptoms of the pneumonia are low fever, cough, blood-tinged sputum, asthma. Large numbers of worms may give rise to allergic symptoms. Eosionophilia is generally present. These clinical manifestation is also called Loeffler’s syndrome.
2. The intestinal phase of the adults. The presence of a few adult worms in the lumen of the small intestine usually produces no symptoms, but may give rise to vague abdominal pains or intermittent colic, especially in children. A heavy worm burden can result in malnutrition. More serious manifestations have been observed. Wandering adults may block the appendical lumen or the common bile duct and even perforate the intestinal wall. Thus complications of ascariasis, such as intestinal obstruction, appendicitis, biliary ascariasis, perforation of the intestine, cholecystitis, pancreatitis and peritonitis, etc., may occur, in which biliary ascariasis is the most common complication.

Diagnosis

¨ The symptoms and signs are for reference only. The confirmative diagnosis depends on the recovery and identification of the worm or its egg.

1. Ascaris pneumonitis: examination of sputum for Ascaris larvae is sometimes successful.

2. Intestinal ascariasis: feces are examined for the ascaris eggs.

(1) direct fecal film: it is simple and effective. The eggs are easily found using this way due to a large number of the female oviposition, approximately 240,000 eggs per worm per day. So this method is the first choice.
(2) brine-floatation method:

(3) recovery of adult worms: when adults or adolescents are found in feces or vomit and tissues and organs from the human infected with ascarids , the diagnosis may be defined.

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articel : Trichuris trichiura

Morphology

Adult: the worm looks like a buggy whip, the anterior 3/5 is slender and the posterior 2/5 is thick. It is pinkish gray in color. The female worm is 3-5 cm in length and has a long slender esophageal region. The male is smaller than the female and has a curved tail. The reproductive organs of male and female are all double tubule.

Egg: it is barrel or spindle in shape and 50 x 20µm in size. It is brownish and has a translucent polar plug at either ends. The content of the egg is an undeveloped cell


Life cycle

Female releases 1000 to 7000 unembryonated double-plugged eggs each day.Eggs are deposited onto warm, moist shady soil where they embryonate to the L1 stage.

The emrionate egg is the infective stage and is ingested. L1 hatch from the egg in the small intestine and burrow into the mucosa where they are. Adults migrate to the ileocecal and colon regions. Adult worm is inserted into the wall of the intestine penetrating through the mucosa and into the submucosa where blood is ingested.

Pathogenesis:

1.Light infection: Asymptomatic

2.Middle infection: Clinical manifestations are usually abdominal pain, anorexia, diarrhea, constipation .

3. Heavy infection: Bloody diarrhea, emaciation, prolapse of the anus may occur.

Diagnosis:

Discover the eggs in feces by saturated brine flotation method or direct fecal smear.

Treatment and prevention: Same as those of ascariasis

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article : Enterobius vermicularis (Pinworm)

The pinworms are one of the most common intestinal nematodes. The adult worms inhabit the cecum and colon. Right after mating, the male dies. Therefore, the male worms are rarely seen. The female worms migrate out the anus depositing eggs on the perianal skin. Humans get this infection by mouth and by autoinfection.


Morfology

Adults: The adults look like a pin and are white in color. The female worm measures about 8 to 13 mm in size and is fusiform in shape. The male adult is only 2-5mm. The tail of a male is curved. They die right after mating, thus males are rarely seen. The anterior end tapers and is flanked on each side by cuticular extensions called “ cephalic alae”. The esophagus is slender, terminating in a prominent posterior bulb , which is called esophageal bulb. The cephalic alae and esophageal bulb are important in identification of the species.

Egg: 50 to 60m by 25 µm, persimmon seed-like, colorless and transparent, thick and asymmetric shell, content is a larva.Anal smear showing large numbers of Enterobius eggs under the lower power. In the background are also two Ascaris eggs.

Life cycle

site of inhabitation: cecum and colon , infective stage: embryonated egg and infective route: by mouth, without intermediate host and reservoir host, life span of female adults: 1-2 months.

About one-third of pinworm-infected persons are asymptomatic, The adult worms may cause slight irritation of the intestinal mucosa.

Major symptom is anal pruritus, which associates with the nocturnal migration of the gravid females from the anus and deposition of eggs in the perianal folds of the skin. Restlessness, nervousness, and irritability, probably resulting from poor sleep associated with anal pruritus,. In young girls, migration of the worms may produce vaginitis and salpingitis or granuloma of the peritoneal cavity.

Diagnosis

Diagnosis depends on recovery of the characteristic eggs. The eggs and the female adults can be removed from the folds of the skin in the perianal regions by the use of the cellophane tape method. The examination should be made in the morning, before the patient has washed or defecated.

Treatment & Prevention

Since the life span of the pinworm is less than two months, the major problem is reinfection. Albendazole is the drug of choice. Repeated retreatment may be necessary for a radical cure.

Prevention:
1. treat the patients and carriers
2. individual health
3. public health
4. health education and hygienic habits

Epidemiology

Geographical distribution—cosmopolitan in temperate zones with about 30 to 50% of the population infected. It is more common in white than colored people and more prevalent in children than adults. Enterobiasis is most common where people live under crowded conditions such as orphanages, kindergartens, and large families.

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article : More Know about Hookworm

The hookworms cause hookworm disease, which is one of the five major parasitic disease in China(malaria, shistosomiasis, filariasis, kala- azar and hookworm disease). At least two species of hookworms infect man, Necator americanus and Ancylostoma duodenale. They live in small intestine.

Morfology

Adults: They look like an odd piece thread and are about 1cm. They are white or light pinkish when living. ♀is slightly larger than♂.The male’s posterior end is expanded to form a copulatory bursa.

Eggs: 60×40 µm in size, oval in shape, shell is thin and colorless.

Content is 2-8cells.

The Morphological Differences between Two species of Hookworms

____________________________________________________

A. duodenale N. americanus

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Size larger smaller

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Shape single curve, looks like C double curves, looks like S

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Mouth 2 pairs of ventral teeth 1peir of ventral cutting plates

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Copulatory circle in shape oval in shape

Bursa (a top view) (a top view)

____________________________________________________________

Copulatory 1pair with separate 1pair of which unite to form

spicule endings a terminal hooklet

_______________________________________________________

caudal spine present no

_______________________________________________________

vulva position post-equatorial pre-equatorial

_______________________________________________________

Lyfe cycle

1. Final host: man

2. Inf. Stage: Larva 3 or filariform larva

3. Inf. Route: by skin

4. Food: blood and tissue fluid

5. Site of inhabitation: small intestine

6. Life span: Ad 15years, Na 3-7years

7. Blood-lung migration:

skin, cavum, right heart, lungs

nPathogenesis & Clinical manifestation

Larval migration

(1) Dermatitis, known as "ground itch" or "stool poison".The larvae penetrating the skin cause allergic reaction, petechiae 0r papule with itching and burning sensation. Scratching leads to secondary infection.

(2) pneumonitis (allergic reaction), Loeffier's syndrome: cough, asthma, low fever, biood-tinged sputum or hemoptysis, chest-pain, inflammation shadows in lungs under X-ray. These manifestations go on about 2 weeks.

Adults in small intestine

(1) Epigastric pain as that of a duodenal ulcer.

(2) A large worm burden results in microcytic hypochromatic anemia (character manifestation). The symptoms are lassitude, edema, palpitation of the heart. In severe case, death may result from cardiac failure or physical exhaustion.

(3) Allotriophagy (orpica) is due to the lack of trace element iron (4) Amenorrhea, sterility, abortionmay take place in women.

(5) Gastrointestinal bleeding

(6) Infantile hookworm disease

Diagnosis

Criterion: 1. hemoglobin is lower than 120g/L in man,
110g/L in woman. 2. find hookworm egg

Method:

1. saturated brine flotation technique

2. direct fecal smear

3. culture of larvae

Treatment

1. Albendazole
2. Mebedazole

Epidemiology

worldwide distribution. 22-26℃ is the optimal temperature for Ancylostoma duodenale development, Ancylostoma duodenale mainly prevalent in north of China. 31-35℃ is suitable for Necator americanus, it is mainly prevalent in south of China

Prevention

Unified measures:
1. sanitary disposal of night soil,
2. individual protection,
3. health education,
4. cultivate hygienic habits,
5. treat the patients and carriers.

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Magister Research

Study of methane production by methanogenic consortium bacteria with a substrate of Jatropha curcas seed pressed cake

Yati Supriatin, I Nyoman P. Aryantha, Siti Khadijah Chaerun

School of Life Sciences and Technology

Institut Teknologi Bandung

ABSTRACT

The study of biogas production with seed pressed cake of Jatropha curcas as a substrate in a 2.5 L reactor scale aims to know the optimum condition to produce biogas (methane). The research was begun by screening the inoculum source from cow dung, dam sediment, and cow rumen liquid. The potency of single and consortium bacteria to produce biogas with the highest methane concentration was evaluated. After the best microbial agents was determined, the next step was the substrate composition using jatropha seed pressed cake with C/N ratio of 12.6 and jatropha seed pressed cake added with tapioca waste with total C/N ratio of 15. Three variations of inoculums concentrations (5%, 10%, and 20%) with four times repetitions were investigated. The system was inoculated with 30 day old inoculum and observed for 70 days by measuring several physical, chemical, and biological parameters in a batch system reactor. The result shows that natural consortium from Jatiluhur dam sediment produces the highest methane gas concentration. Jatropha seed pressed cake with C/N ratio 12.6 and inoculums concentration of 20% gave the best result. Biogas was produced from 14^th day and then increased until 70^th day with total production of 2.822,25 mL (average 40.31 mL per day). GC analysis showed that methane concentration at day 49^th reached 60.57% and then continued to increase until the 70^th day with a concentration of 74.72%. This value is the highest result compared with other achievements reported in literatures. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus. Biogas was produced the best at a range of pH from 6 - 8. This result can be used as a reference in biogas production with seed pressed cake of J. curcas as a substrate in application scale.

Keywords: biogas, methane, methanogenic bacteria, seed pressed cake, Jatropha curcas.

1. Introduction

The fossil fuel consumption in Indonesia increasingly at several years while production was decreased (Hambali et al, 2006). This problem could be solve with search new alternative energy source. Biogas as gas generated from organic digestion in anaerobic conditions by mixed population of microorganisms is one of alternative energy source, that can be produced from various of wastes. Biogas generally composes of methane (55-65 %), carbondioxide (35-45%), nitrogen (0-3%), hydrogen (0-1%), and hydrogen sulfide (0-1%) (Anunputtikul & Rodtong, 2004). Jatropha seed pressed cake as a bio diesel production waste will be abundant in Indonesia for few years later because developing of bio diesel as one of bio fuel (Hambali et al, 2006). The usage of Jatropha seed pressed cake as substrate to produce biogas had been developed in nicaragua since 1997 (Gubitz, 1997), nevertheless biogas system from Jatropha seed pressed cake have not yet many developed in Indonesia. The aim of this study is to get optimum environment condition in biogas production and the result can be used as a reference in biogas production with Jatropha seed pressed cake as a substrate in application scale.

2. Materials & Method

2.1 Preparation of Substrate

Jatropha seed pressed cake as main substrate in this study were collected from PT Rajawali Nusantara Indonesia Jatitujuh Majalengka. Tapioca waste used as additional substrate was collected from tapioca factory in Tasikmalaya. Total solid (TS), total carbon, total nitrogen, ash, and sulfur content of raw material were determined using standart methods (Oei & Reinhard, 1989).

2.2 Preparation of inoculum source

Inoculum source obtained from cow rumen liquid was collected from animal slaughtering house in Ciroyom, cow dung was collected from dairy cattle ranch in Cimenyan, and Jatiluhur dam sediment deepness 28 metres. Isolation of methanogenic bacteria from 3 inoculum sources have be done to get single methanogenic isolate for compare the biogas production ability with natural consortium bacteria. Isolate screen base on gas production ability (Lay,1994 modified) and than confirm test base on morphology test under fluorescent microscope (Mink & Dugan, 1976). Natural consortium bacteria from three sources kept in a closed container respectively with regular adding a small amount of jatropha seed pressed cake for 14 days and screen base on methane gas production that measured by using gas chromatography at the same time, inoculum source with the higher methane gas production would be kept for 30 days before inoculating the biogas production reactor.

2.3 Biogas production from Jatropha seed pressed cake

The biogas production from Jatropha seed pressed cake was perfomed using the simple-single-state reactor with working volume of 2 liters (L) in batch system. Substrate formulation carried out in two variation that were only Jatropha seed pressed cake with C/N ratio 12.6 and Jatropha seed pressed cake added tapioca waste with mixed C/N ratio 15. Substrate concentration was 10 % (w/v) to water (Hernas, 2007), and inoculum concentration variation was 5%, 10%, 20% (v/v). The biogas fermentation was then operated in four times repetitions at ambient temperature for 70 days by measuring several physical, chemical, and biological parameters.

Change of physical parameters involved odor and texture slurry, flame test. Change of chemical parameters involved COD used bi chromate method (Oei & Reinhard,1989), Measurement of pH used pH meter (Uchida KT-1A), total gas volume used water displacement method (Anunputtikul & Rodtong,2004), and biogas composition used gas chromatography (Shimatdzu, GC8A, Japan) equipped with column Porapak- Q and Mulsife 5A, injector temperature and detector temperature 70⁰C, column temperature 40⁰C, detector type Thermal Conductivity Detector, gas Argon as the carrier gas with speed 30 cc/minute. Change of C/N ratio during fermentation measured by using Khjedahl method for nitrogen content analysis whereas carbon total content analysis by counted difference of dry weight & ash weight (Oei & Reinhard, 1989).

Biological parameters involved change of aerobic and anaerobic bacteria population by using FDA method (Green et al., 2005 modified), bacteria cell enumeration used plate count method (Cappucino & Sherman, 2005), Isolation and identification aerobic and anaerobic dominant culture able bacteria that cultured at seventh day and 70th day used pour plate method and used nutrient agar media (Cappucino & Sherman, 2005). Quantitative Analysis of methanogenic bacteria had be done at seventh day, 30th day and 70th day used viable count method (Lay, 1994 modified).Reactor design showed at figure 1

3. Result & Discussion

3.1 Raw material for biogas production

Jatropha seed pressed cake was bio diesel production waste that produced from Jatropha curcas seed. Some physical and chemical compositions of Jatropha seed pressed cake were analyzed (Table 1).

Table 1 physical and chemical compositions of substrate

No	Composition (% dry weight)	Jatropha seed pressed cake	Tapioca waste
1.	moisture	10.94	12.67
2.	carbon	84.20	77.393
3.	Nitrogen	6.68	0.393
4.	S	4.53	0.0047
5.	TS	92	80.09
6.	Ash	6.7	2.7

3.2 Biogas production from Jatropha seed pressed cake

Result of methanogenic bacteria isolation & screening state was obtained only one isolate bacterium, because of only one isolate that produced, so this research not enables use isolation result as inoculum. Then the research was more developed at usage natural consortium as inoculum. The biggest methane gas production in natural consortium bacteria screening process was achieved by natural consortium bacteria that indigenous in Jatiluhur dam sediment. It happened because Microorganism variety was very height in sediment and it be caused of sediment supports aerobic micro environment forming anaerobic condition (Maier et al., 2003). Methane gas that produced from sediment could be more than cow rumen liquid. According to Madigan et al. (2003) cow rumen liquid had more CO₂ composition that was 65% than CH₄ that only reach 35%, this condition happen because acetotroph methanogenic bacteria in rumen slow growth whereas retention time for the happening of acetate conversion become CH₄ is short time & sintrophyc bacteria fatty acid degrader not play main role in rumen because fatty acid will be adsorb by intestinal wall and enter to circulation system (Madigan et al., 2003).The result of treatment state was shown in figure 2 &3.

the treatment which higher CH₄ gas production was achieved by C/N ratio 12.6 and inoculum concentration 20 %, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2.822,25 mL and the average of production biogas per day 40.31 mL.

In C/N ratio substrate 12.6 the system more balance between non methanogenic bacteria & methanogenic bacteria activity compare in C/N ratio substrate 15. Change of pH during fermentation process was decreased at initial pH 8 became average 4-5, moreover in C/N ratio 15 pH decreased drastically became 2-3, it cause methane formation was obstructed.

When a biogas plant is newly started, the acid formation become active first, reducing the pH, then methanogenic bacteria start using these acid, increasing the pH back to neutral (Fulford, 1988) , but it not happen in C/N ratio 15 because of acid forming to much for methanogenic bacteria could consumption (Burke,2001), finally organic matter in C/N ratio substrate 15 was not convert in CH₄ gas. In this study result that in C/N ratio 12.6 CH₄ gas production at 42^nd day decreased drastically, it caused by accumulating CO₂ gas as result of aerobic bacteria metabolism that achieved 2,4 x 10⁷ CFU/mL sample , but after that CH₄ gas production increased until 70^th day. This result appropriate with result of methanogenic bacteria quantitative analysis that the amount of methanogenic bacteria achieve 5.1 x 10³ cell/mL then continued to increased until 16 x 10³ cell/mL at 70^th day (figure 4).

The result of aerobic & anaerobic bacteria population show that aerobic⁷ CFU/mL sampel while bacteria population achieved 10 anaerobic bacteria population reaches 10⁴ CFU/mL sample. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus.

Conclusion

1. Inoculum that produce highest methane gas production is natural consortium bacteria from Jatiluhur dam sediment with the average of methane gas production that produced is 7.163 % ± 0.689.

2. Substrate that produce highest methane gas is Jatropha seed pressed cake with C/N ratio 12.6 with result 74.72% ± 0.256.

3. Inoculum concentration that produce highest methane gas is inoculum concentration 20%, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2,822,25 mLs and the average of production biogas per day 40.31 mL

Refferences

Anunputtikul, W., & Rodtong, S. (2004 ), The Joint International conference on “ Sustainable Energy and Environment (SEE)”. Laboratory Scale Experiment for Biogas Production from Cassava Tubers. : Hua Hin, Thailand.

Buchanan, R., & Gibbons, E. (1974), Bergey’s Manual of Determinative Bacteriology, 8^th ed., The Williams & Wilkins Co., Inc., USA.

Burke, D.A. (2001), Dairy Waste Anaerobic Digestion Handbook. Environmental Energy Company, Hill Street Olympia.

Cappuccino, J.G., & Sherman, N. (2005), Microbiology a Laboratory Manual, 7^th Ed, The Benjamin/Cummings Publishing Company, Inc. San Francisco

Dubey, S.K. (2005), Microbial ecology of methane emission in rice agroecosystem, Applied Ecology and Environmental Research, 3(2), 1-27. Dalam situs :http//www.ecology .kee.hu/pdf/0302_001027.pdf.

Edwards,T., & McBride,B,C. (1975), New Method for the Isolation and Identification of Metanogenic Bacteria. Applied Microbiology. American Society for Microbiology. 29 (4). 540-545

Fulford, D. (1988), Running A Biogas Programme: A handbook.ITDG Publishing., London.

Green, V. S., Stott, D.E., & Diack, M. (2005), Assay for fluorescein diaceate hydrolitic activity : optimization for soil sampes, Soil Biol. & Biochem., 38, 693 – 701

Gubitz, M.G., Mittelbach., & Trabi, M. (1997), Biofuel and Industrial Products from Jatropha curcas. Developed from the Symposium Jatropha curcas 97, Managua, Nicaragua.

Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., & Williams, S.T. (1994), Bergey’s Manual of Determinative Bacteriology, 9^th ed., The Williams & Wilkins Co., Inc., USA.

Hernas,T., Kelompok Riset Biodiesel ITB. (2007), Biogas dari Bungkil Jarak Pagar , inovasi terbaru dari teknik Kimia ITB. ITB News.

Hambali, E., Muzdalifah, S., Sulistyanto, G., & Lesmana, T.(2006), Diversifikasi Produk olahan Jarak Pagar & Kaitannya dengan CSR Perusahaan Swasta di Indonesia. Eka Cipta Foundation, Bogor.

Lay, Bibiana.W. (1994), Analisis Mikroba di Laboratorium. P.T Raja Grafindo Persada, Jakarta.

Mink, W.R., & Dugan, R.P. (1976), Tentative Identification of Metanogenic Bacteria by Fluorescence Microscopy. Applied Microbiology. American Society for Microbiology. 33 (3). 713-717.

Madigan, M.T., Martinko, J.M.& Parker, J. (2003), Biology of Microorganisms, 9^th Ed, Pearson Education, Inc., USA.

Maier, R.M., Pepper, I.L., & Gerba, C.P. (2003), Environmental Microbiology, Academic Press, San Diego.

Oei, B. L., & Reinhard, J. (1989), Metode Analisa pada Fermentasi Anaerob. Pusat Antar Universitas Bioteknologi Institut Teknologi Bandung.

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