Isolation and Detection of Escherichia Coli Trimethoprim Resistance

Gene from Layer Chickens in East Java Province, Indonesia by

Polymerase Chain Reaction

Emy Koestanti Sabdoningrum

, Sri Hidanah

, Sri Chusniati

, Wiwik Misaco

Retno Sri Wahyuni

Laras Retno Kinasih Harianto

Department of Animal Husbandry, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia

Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia

Department of Clinic, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia

Department of Veterinary Basic Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia

Student, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia

Keywords: Escherichia coli, Trimethoprim resistance gene, Layer chicken, Polymerase Chain Reaction.

Abstract: Aim: The aim of this research was to detect the presence of a gene which is responsible for Escherichia coli

trimethoprim resistance (TEM gene) using Polymerase Chain Reaction from layer chickens in East Java

Province, Indonesia. Methods: Purposive sampling of 60 infundibulums has been done in laying chicken

farms in those indicating Colibacillosis from 6 districts in East Java Province, Indonesia including Sidoarjo,

Mojokerto, Jombang, Kediri, Bojonegoro and Blitar. Ten samples were collected from each district.

Samples were isolated using Eosin Methylene Blue Agar and five biochemical tests of Sulfide Indol

Motility (SIM), Simons Citrate Agar (SCA), Triple Sugar Iron Agar (TSIA), Urea Agar (UA) and Sugar

Test were used for bacteria identification. The DNA was then isolated from Escherichia coli-positive

samples prior to Polymerase Chain Reaction (PCR) amplification to investigate TEM gene that indicated

trimethoprim antibiotic resistance. Result: Bacteriological tests showed that 30 of 60 samples (50%) were E.

coli positive. Ten of those were from Blitar, 10 from Bojonegoro, 5 from Sidoarjo, and 5 from Jombang.

Two samples from Kediri and Mojokerto districts were negative. From PCR amplification showed that 28

of 30 samples were negative due to TEM gene, and there were 2 positive samples shown from Bojonegoro

and Blitar. This indicated that 6.66% of total samples were positive containing TEM gene. Conclusion:

Based on bacteriological examination 50% of samples from six districts in East Java, Indonesia were E. coli

positive, and 6.66% were trimethoprim resistant based on occurrence of TEM gen in PCR amplification.

1 INTRODUCTION

Avian colibacillosis is an infectious avian disease of

caused by Escherichia coli infection and is

responsible for significant economic losses in the

poultry industry (Matsuda et al., 2010). Avian

Pathogens Escherichia coli (APEC) are increasingly

encountered in the field and its relationship to

various conditions of the disease, either as a primary

pathogen or as a secondary pathogen and may be

infectious to humans or zoonotic. Chicken

colibacillosis is characterized in acute form by

septicemia resulting in death and subacute form by

pericarditis, air sacculitis and perihepatitis. Avian

colibacillosis has been recognized as a major

contagious disease of all ages. Avian Pathogenic

Escherichia coli (APEC), spreads to various internal

organs and causes colibacillosis characterized by

systemic fatal disease. The diseases that are infected

by E. coli enter the host through ingestion or

inhalation, after which it trans-locates across

mucosal layers, then colonizes in other tissue via

bloodstream, it can cause air sacculitis, enteritis,

arthritis, panoptalmitis, reproduction genital

infections, bursitis stenalis (Krisnaningsih, 2005),

and in layers can cause salphingitis, omphalitis,

misshapen, pedunculated eggs and egg peritonitis

(Anyanwu, 2014).

Treatment with many antibiotics causes

resistance. In the use of antibiotics in APEC it is

292

Koestanti Sabdoningrum, E., Hidanah, S., Chusniati, S., Misaco, W., Sri Wahyuni, R. and Retno Kinasih Harianto, L.

Isolation and Detection of Escherichia Coli Trimethoprim Resistance Gene from Layer Chickens in East Java Province, Indonesia by Polymerase Chain Reaction.

DOI: 10.5220/0007541602920300

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 292-300

ISBN: 978-989-758-348-3

important to note the different sensitivity of E. coli

serotype because some serotypes were resistant to

some antibiotics. The development of resistance

properties of E. coli bacteria is a serious problem

today especially related to the treatment and

handling of some diseases caused by E. coli, so it is

necessary to do a safe antibiotic replacement. The

use of inappropriate antibiotics with no benefit to

therapy is a feed stimulator or growth promoter for

livestock and poultry is one cause of loss of

antibiotic effectiveness. It can cause disruption of

normal microbial ecology balance and eliminate a

group of sensitive bacteria while a group of resistant

bacteria will grow and well developed becomes a

pathogenic population (Dibner & Richards, 2005).

Trimethoprim is a potent broad-spectrum

antibacterial agent with a slow bactericidal action

against susceptible bacterial infections (gram-

negative and gram-positive). It acts by inhibiting the

enzyme dihydrofolate reductase, which is

responsible for the conversion of folate to folinate

and reduces their pools of folinic acid that is

required as a co-factor (Papich, 2016). Nowadays,

there is widespread antibiotic resistance in the

poultry industry and can lead to reconsideration of

antibiotic use, especially trimethoprim which has

been widely used primarily by combining with the

sulfonamide group. Bacteria may become resistant

to trimethoprim by the following: reduced bacterial

uptake, alterations or mutations in dihydrofolate

reductase, overproduction of dihydrofolate reductase

(Kester, et al., 2012).

The aim of this research was to detect the

presence of trimethoprim antibiotic resistance gene

in isolated samples taken from layer chicken farms

from East Java Province, Indonesia using the latest

method of matching genes using PCR.

2 MATERIALS AND METHODS

The survey areas were the layer chicken farms

owned by breeder farmers in East Java Province,

Indonesia, at Sidoarjo, Mojokerto, Jombang, Kediri,

Bojonegoro and Blitar districts.

2.1 Sampling Areas

Ten samples of infundibulum were taken from each

district indicating colibacillosis and the total number

of samples was 60 samples.

2.2 Isolation of Samples

Samples were taken from layer chicken’s

infundibulum and then isolated using Eosin

Methylene Blue Agar medium and five biochemical

tests of Sulfide Indol Motility (SIM), Simons Citrate

Agar (SCA), Triple Sugar Iron Agar (TSIA), Urea

Agar (UA) and Sugar Test were used for bacteria

identification. Positive results of E. coli gained from

biochemical tests showed that SIM had a cloudy

streak area and a red ring on top of the medium,

TSIA medium showed A/A, H

S (-), and gas (+),

Urea Agar changed colour from pink to orange. On

Sugar Test almost all of the sugar was fermented

except mannitol, and for SCA Medium did not

change into any colour.

2.3 DNA Isolation

After biochemical test showed positive results, E.

coli was inoculated in Nutrient Agar to avoid any

disturbing colours as PCR processes take place from

EMBA medium. These were the following steps for

the extraction of DNA:

Took 20 μl QIAGEN Protease (or proteinase K)

using pipet into 1,5 ml micro-centrifuge tube. Add

180 μl ATL Buffer, then add 2-3 bacteria colony

from Nutrient Agar and 5 μl Lysozim. Incubation at

60°C for 30 minutes. Add 200 μl AL buffer into the

sample, then vortex for 15 seconds. Add 200 μl

ethanol 96% and mix up with vortex for 15 seconds,

then spin down. Put into QIAamp Mini spin column

(2 ml collection tube) compound from step before.

Centrifuge at 8,000 rpm for 1 minute. Throw 2 ml

filtrate from collection tube and then replaced tube

with the new one (new 2 ml collection tube). Add

500 μl AW1 Buffer, then centrifuge at 8,000 rpm for

1 minute. Throw 2 ml filtrate from collection tube

and then replace tube with the new one (new 2 ml

collection tube). Add 500 μl AW2 Buffer, then

centrifuge at 13,000 rpm for 3 minutes. Throw 2 ml

filtrate from collection tube and then replace tube

with the new 2 ml collection tube. Centrifuge again

at 13,000 rpm for 1 minute. Move QIAamp Mini

spin column to 1.5 ml micro-centrifuge tube. Add 50

μl AE Buffer or distilled water. Incubation at room

temperature (15-25°C) for 1 minute, then centrifuge

at 8,000 rpm for 1 minute. Then obtain 50 μl DNA

template.

Isolation and Detection of Escherichia Coli Trimethoprim Resistance Gene from Layer Chickens in East Java Province, Indonesia by

Polymerase Chain Reaction

293

2.4 Polymerase Chain Reaction (PCR) and

Electrophoresis

Materials for Polymerase Chain Reaction running

were as follows: 20 μl QIAGEN Protease

(proteinase K), 180 μl Buffer ATL, 5 μl Lysozyme,

200 μl buffer AL, 200 μl ethanol 96%, 500 μl Buffer

AW1, 500 μl Buffer AW2, 50 μl Buffer AE, 2x PCR

Master mix (Intron), 1µl (50Pmol/µl) of TEM F3

(the sequence of primer pair is GTA TCC GCT CAT

GGA GAC AAT AAC CCT G) and TEM R3 (the

sequence of primer pair is CCA ATG CTT AAT

CAG TGG AGG CAC C) primer, and 5µl of DNA

template, agarose gel 1.5%.

Amplification of bacterial DNA was performed

with a volume of 20 µl as follows: 12.5 µl of 2X

master mix (Intron), 0.5µl of distilled water, 1 µl of

TEM F3 primer, 1 µl of TEM R3 primer, and 5 µl of

DNA template. Polymerase chain reaction steps are

Pre-Denaturation at 94

C for 5 minutes, then

Denaturation at 94

C for 1 minute; Annealing at

C for 30 seconds for 30 times; Extension at 72

for 1 minute, these 3 steps are repeated for 35 cycles

and last Final Extension at 72

C for 5 minutes.

PCR products are electrophoresed on a 1.5%

agarose gel then running for 30 minutes. A digital

image of the gel is captured in a computer, and the

amplification patterns were evaluated by visual

examination of inverted gel pictures.

3 RESULTS

The survey showed that layer chickens in Sidoarjo,

Mojokerto, Jombang, Kediri, Bojonegoro and Blitar

district indicating colibacillosis showed by number

of cases found in the field and from the checking of

post-mortem showed symptoms of colibacillosis.

There were 10 layer chickens obtained from each

district. The bacteriological test showed that 10

samples from Blitar, 10 samples from Bojonegoro, 5

samples from Sidoarjo, and 5 samples from

Jombang were E. coli positive. Samples from Kediri

and Mojokerto were negative.

Table 1 : Result of biochemical test for E. coli

No. District

Samples

Amount

Result

Positive Negative

1. Sidoar

o 10 5 5

2. Mo

okerto 10 0 10

3. Jomban

10 5 5

4. Kediri 10 0 10

5. Bo

one

oro 10 10 0

6. Blitar 10 10 0

Table 2. Detection of TEM gene in samples from East

Java Province, Indonesia

No. District

Samples

Amount

Result

Positive Negative

1. Sidoar

o5 0 5

2. Mo

okerto 0 0 0

3. Jomban

50 5

4. Kediri 0 0 0

5. Bo

one

oro 10 1 9

6. Blitar 10 1 9

Table 3 : Results of isolation and identification for E. coli using bacteriological tests

Area

Number of

Sample

EMBA SIM SCA TSIA G L S Malt Man

Sidoarjo

1 - - - - - - - - -

2 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

3 - - - - - - - - -

4 - - - - - - - - -

5 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

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6 - - - - - - - - -

7 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

8 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

9 - - - - - - - - -

10 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

Mojokerto

1 - - - - - - - - -

2 - - - - - - - - -

3 - - - - - - - - -

4 - - - - - - - - -

5 - - - - - - - - -

6 - - - - - - - - -

7 - - - - - - - - -

8 - - - - - - - - -

9 - - - - - - - - -

10 - - - - - - - - -

Jombang

1 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

2 - - - - - - - - -

3 - - - - - - - - -

4 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

5 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

6 - - - - - - - - -

7 - - - - - - - - -

8 - - - - - - - - -

9 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

10 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

Isolation and Detection of Escherichia Coli Trimethoprim Resistance Gene from Layer Chickens in East Java Province, Indonesia by

Polymerase Chain Reaction

295

Kediri

1 - - - - - - - - -

2 - - - - - - - - -

3 - - - - - - - - -

4 - - - - - - - - -

5 - - - - - - - - -

6 - - - - - - - - -

7 - - - - - - - - -

8 - - - - - - - - -

9 - - - - - - - - -

10 - - - - - - - - -

Bojonegoro

1 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

2 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

3 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

4 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

5 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

6 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

7 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

8 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

9 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

10 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

Blitar

1 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

2 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

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3 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

4 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

5 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

6 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

7 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

8 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

9 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

10 + +/motile -

A/A, H

S (-) gas

(+)

+ + + + -

4 DISCUSSION

Colony of E. coli on EMBA medium showed

metallic green colour with uniform colony, with

circular colony shape, rough surface, low convex

elevation, and erose edges. EMBA medium is a

selective and differential medium for gram-negative

bacteria coliform containing eosin and methylene

blue which is an indicator of pH and positive gram

bacterial inhibitors. In addition, EMBA media can

distinguish coliform bacteria as normal flora and E.

coli in the presence of lactose and sucrose indicator.

All sugar tests resulted in yellow colour of the

medium due to the fermentation of sugar which

caused the pH of the media to turn into acid. In this

research, the result shows that no changing colour of

SCA medium indicated that E. coli bacteria did not

use citrate as carbon sources.

Figure 1 : Colony of E. coli on EMBA medium

Isolation and Detection of Escherichia Coli Trimethoprim Resistance Gene from Layer Chickens in East Java Province, Indonesia by

Polymerase Chain Reaction

297

In the SIM media, indol test aims to identify the

ability of bacteria to produce indole by using

tryptophanase enzyme (Leboffe, 2011). In this

research, observations on the SIM media found a

cloud clump around the streak area. This prompts

the movement of bacteria that grow around the

streak area. The movement of the bacteria due to

semisolid media (motility test) is designed by

reducing the concentration of agar to the media that

is about 0.4% on the medium which is only

sufficient to maintain its shape while allowing the

movement of bacteria (Leboffe, 2011).

Urease test is useful for identifying organisms

capable of hydrolyzing urea which can produce

ammonia and carbon dioxide, especially to know if

the microorganisms have urease enzyme or not.

Urease is a constitutive enzyme that hydrolyzes urea

into carbon dioxide and ammonia. In this research,

Urea Agar used was not pink, but a little orange in

colour and from the observational result the agar

media did not change in colour.

In this research, results from observations for the

TSIA test showed A/A with negative H

S and

presence of gas. The yellow colour of the entire

medium is due to the fermentation of carbohydrates

and will bring up the gas as a gap in the media or

will lift the agar from the bottom of the tube

(Leboffe, 2011).

Using the PCR method can give faster, cheaper

and more efficient results. If using another method

such as dilution with MIC (Minimum Inhibitory

Concentration) and MBC (Minimum Bacteriosid

concentration) that takes longer time to see the

result. Based on the results of resistance test

antibiotic trimethoprim using PCR it can be seen

that the results of the Bojonegoro and Blitar districts

show positive antibiotic resistance trimethoprim.

This is indicated by the emergence of bands on

electrophoresis results at 861 bp. The presence of

bands that appear on the results indicates that there

are bacteria that have gene matches with the

existing primary pieces. This means that the bacteria

have adapted from the previous environment so that

it has a gene that can withstand the sensitivity of the

trimethoprim antibiotic that matches the existing

primer. With the emergence of this band, it can be

concluded that bacteria found in Bojonegoro and

Blitar districts have genetic compatibility with the

primary TEM gene, which indicates antibiotic

resistance to trimethoprim in Escherichia coli.

The resistance rate of trimethoprim among

Escherichia coli from layer chickens in East Java,

Indonesia was 6.66% based on the result of this

research. The resistance occurs because bacteria

produce enzyme decomposers antibiotics so that

antibiotics become inactive. These bacteria encode

genes that produce enzymes that break down

antibiotic molecules before they kill bacteria. An

example is the lactamase beta enzyme, this enzyme

will describe the beta structure of lactam in

antibiotics, so antibiotics become inactive again and

cannot kill bacteria. It reduces the accumulation of

C- E4 E5 E6 E7 M

Figure 3 : Trimethoprim Resistance by Polymerase Chain Reaction Result

Explanation:

Target band 861 bp, 2 positive

samples

M : Marker DNA 100bp

C- : Negative control

E4 : Sidoarjo

E5 : Bojonegoro

E6 : Jombang

E7 : Blitar

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intracellular antibiotics by decreasing permeability

and/or enhancing the active efflux of antibiotics.

The efflux mechanism occurs when a resistant gene

encodes a protein that actively pushes antibiotics out

of a bacterial cell, resulting in low levels of

antibiotics in the cell and inability to kill bacteria.

5 CONCLUSION

Based on biochemical examination, 50% of samples

from six districts in East Java, Indonesia were

Escherichia coli positive, and 6.66% were

trimethoprim resistant.

REFERENCES

Anyanwu, M. U, et. al. 2014. Case Report of

Misdiagnosis of Avian ColibacillosisIn Laying Birds.

Animal Research International (2014) 11(2): 1998-

2003.

Barnes HJ and Yoder HW. 1984. Disease of Poultry. 8

ed. Iowa State University Press. 692-708.

Bettelheim K. A and L. Beutin, 2003. Rapid

laboratory identification and characterization of

verocytotoxigenic (Shiga toxin producing)

Escherichia coli (VTEC/STEC). Journal of Applied

Microbiology. 95: 2: 205-217

Bettelheim KA, 1996. Enterohaemorrhagic Escherichia

coli: a new problem, an old group of organisms. Aust

Vet J 73:20-26

Bettelheim KA, Beutin L, 2003. Rapid Laboratory

Identification and Characterization of

Verocytotoxigenic (Shiga toxin producing )

Escherichia coli (VTEC/STEC). J App Microbiol

95:205-217

Beutin L, Montenegro MA, Ørskov I, Ørskov F, Prada J,

Zimmermann S, Stephan R, 1989. Close association

of verocytotoxin (Shiga-like toxin) production with

enterohemolysin production in strains of Escherichia

coli. J Clin Microbiol 27:2559-2564

Brooks GF, Butel JS, Morse SA. 2005. Medical

Microbiology. McGraw-Hills Companies Inc.

Dibner, J. J & J. D. Richards. 2005. Antibiotics Growth

Promoters in Agriculture: history and mode of action.

Poult. Sci. 84:634-643

Elfahmi. 2006. Phytochemical and biosynthetic studies of

Lignans, with a focus on Indonesian medicinal plants

(dissertation). University of Groningen. 2006.

Gordon, R.F.1997. Poultry Disease. Isted. Bailliere

Tindall and Cox. London.

Gross, T., J. Faull., S. Ketteridge., D. Springham. 1995.

Introductory Microbiology. Chapman and Hall. Great

Britain. 51.

Harborne, J. 1987. Metode Fitokimia, Edisi Kedua. ITB.

Bandung.

Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K,

Yokoyama K, Han CG, Ohtsubo E, Nakayama K,

Murata T, Tanaka M, Tobe T, Iida T, Takami H,

Honda T, Sasakawa C, Ogasawara N, Yasunaga T,

Kuhara S, Shiba T, Hattori M, Shinagawa H, 2001.

Complete genome sequence of enterohemorrhagic

Escherichia coli O157:H7 and genomic comparison

with a laboratory strain K-12. DNA Res. 8:11-22

Kardinan A. dan Kusuma FR. 2004. Meniran penambah

daya tahan tubuh alami. Jakarta: Agromedia Pustaka,

2004; 6-18.

Kester, Mark., K.D. Karpa, K.E. Vrana. 2012. Elsevier’s

Integrated Review: Pharmacology. Second edition.

Philadelphia. Book AID International. Sabre

Foundation.

Krisnaningsih, M.M.F., Widya A., M. H. Wibowo. 2005.

UjiSensitivitasIsolatEscherichia coli Patogen pada

Ayam Terhadap Beberapa Jenis Antibiotik. J. Sain

Vet. Vol. 1.

Leboffe, M. and Pierce, B. (2011) A Photographic Atlas

for the Microbiology Laboratory. 4th Edition, Morton,

Englewood.

Ma’at, S.1997. Phylanthus niruri L Sebagai

Immunostimulator Pada Mencit. Disertasi Program

Sarjana. Universitas Airlangga. Surabaya. Hal: 13-

174.

Madigan, M.T , Martinko, M.J , Parker. J. 2002. Brock

Biology of Microorganism. 10

Edition. Practice

Hall. USA. 375-378

Mathivanan R, Edwin SC, Amutha R and Viswanathan K.

2006. Panchagavya and Andrographis Panicuata as

Alternative to Antibiotic Growth Promoter on Broiler

Production and Carcass Characteristic. India.

Departement of Poultry Science, Veterinary College

and Research Institute. Namakkal-637001.

Matsuda, K., Atul A. C., John Hwa L. 2010. Avian

Colibacillosis caused by an intestinal pathogenic

Escherichia coli isolate from calf diarrhea. Research

in Veterinary Science 89: 150-152.

Matsuda, K., Atul A. C., John Hwa L. 2010. Avian

Colibacillosis caused by an intestinal pathogenic

Escherichia coli isolate from calf diarrhea. Research

in Veterinary Science 89: 150-152.

Nataro JP, Kaper JB, 1998. Diarrheagenic Escherichia

coli. Clin Microbiol Rev 11:142-201

Pelczar, J. Michael dan chan, E.C.S. 1988. Dasar-dasar

Mikrobiologi 2. Penerbit UI Press. Jakarta.

Robinson, T. 1995. Kandungan Organik Tumbuhan

Tinggi. Institut Teknologi Bandung, Bandung.

Rohimah. 1997. Identifikasi flavonoid yang Memiliki

Antifungal dari Damar (Hope mangarawan) dan

Shoren eptosula. [Tesis]. FMIPA-IPB. Bogor.

Sunarno. 2007. Efek Phyllanthus Niruri L Pada

Prosentase Neutrofil, Koloni Bakteri Limpa, dan

Histopatologi Hepar Balb/C yang Diinfeksi

Salmonella [Tesis]. Universitas Diponegoro

Semarang.

Tizard, IR. 1996. Veterinary Immunology an Introduction.

Fifth Edition, WB Sounders Company, a Division of

Harcourt Brace and Company. The Curtis Center

Isolation and Detection of Escherichia Coli Trimethoprim Resistance Gene from Layer Chickens in East Java Province, Indonesia by

Polymerase Chain Reaction

299

Independence Square West, Philadelphia,

Pennsylvania.

Tjandrawinata, R.R., S. Maat and D. Noviarny, 2005.

Effect of standardized Phyllanthus niruri extract on

changes in immunologic parameters: correlation

between preclinical and clinical studies. Medika

XXXI (6): 367-371.

Tjay, dan Raharja. 2002, Obat-Obat Penting: Khasiat

Penggunaan dan Efek-Efek Sampingnya, Edisi

Kelima, Cetakan Kedua, Penerbit PT. Alex Media

Komputindo, Jakarta.

Wahyuningsih. 2008. Pengaruh Pemberian Ekstrak Herba

Meniran (Phyllanthus Niruri L.) Terhadap Penurunan

Kadar Asam Urat Darah Tikus Putih Jantan

Hiperurisemia. Lampung.

Widayati, P. 2008. Efek Ekstrak Etanol Herba Meniran

(Phyllanthus niruri L.) Terhadap Penurunan Kadar

Asam Urat Mencit Putih Jantan Galur Balb-C

Hiperurisemia [Skripsi]. Universitas Muhammadiyah

Surakarta.

Wijayakusuma dan Hembing H.M. 2003. Ramuan

Tradisional Untuk Pengobatan Darah Tinggi. Penebar

Swadaya. Jakarta. 64-65.

Willey J.M, Sherwood L.M, Woolverten C.J. 2009.

Prescott’s Principle of Microbiology. McGraw-Hill

Higher Education. New York.

Zhu P, S. Li, C-M. Tang, D. Shelton. 2008. Sensitive and

Rapid Detection of Escherichia coli O157:H7 by

Coupling of Immunomagnetic Separation with

Fluorescence Immunoassay. American Society for

Microbiology

ICPS 2018 - 2nd International Conference Postgraduate School

300