Detection Of Virulence Associated Genes


A total of 631 different samples (milk, faeces, brain and Cerebrospinal fluid) were collected from 268 diseased farm animals and emergency slaughtered animals (80 sheep, 60goat, 75 cattle and 53 buffaloes) from Dakahlia Governorate in 2014 and 2015. The samples were tested for presence of L. monocytogenes by isolation and biochemical identification. 
The results revealed that L. monocytogenes was presented in diseased and emergency slaughtered sheep, goat, cattle and buffaloes in ratio of (36.5%), (28.67%), (24.48%), (17.4%) respectively. Using multiplex PCR technique, detected the presence of virulence-associated genes Detection Of Virulence Associated Genes In ... 

INTRODUCTION

L. monocytogenes is a food borne pathogen of major concern with regard to public and animal health. This bacterium affects a wide range of mammalian species, most commonly humans and domestic ruminants Four key of L. monocytogenes virulence genes (i.e plcA, hly, actA, and plcB) that are critical for the intracellular life cycle Ward et al., (2004). The hemolysin (listeriolysin O), two distinct phospholipases, a protein (ActA), several internalins, and others had been identified and extensively characterized at the molecular and cell biologic levels 

Kathariou S (2002).

This study was done to throw spot light on molecular characterization of L. monocytogenes infection in some farm animals including sheep, goat, cattle and buffaloes in El-Dakahlia governerate. 
The namely inlA, inlC, inlJ. plcA, actA, hlyA and jap, inlB and plcB genes for L. monocytogenes were chosen as target genes throughout this study. 

MATERIAL AND METHODS


A


Isolation of L. Monocytogenes:

The technique recommended by United Stated Department of Agriculture (USDA), Food Safety and Inspection Service "FSIS" (1989) and FAO (1992) was adapted. 
Twenty five gm. from each brain-faeces sample were aseptically weighted and homogenized in primary selective Listeria Enrichment Broth, University of Vermont Medium provided from Biolife (LEBUVMI) and incubated at 30°C for 24 hours then 0.1 ml of incubated (LEBUVM1) will be transferred to 10 ml (LEBUVMII) and incubated at 33-37°C after 24 hours of incubation a loopful from enrichment culture UVMII broth were streaked onto PALCAM agar plates (oxoid CM,877) containing selective supplement (oxoid SR150), then incubated at 35-37°C for 24-48 hours (Jemmi and Keusch, 1994). 
Milk samples and C.S.F.: (FDA, Lovett et.al., 1987): twenty five ml. of milk were added to 225ml. of Enrichment Broth Modified tryptone soya broth containing 0.6% yeast extract, Nalidixic cid 40mg/l, Acriflavine hydrochloride 15mg/l and incubated at 30°C for 48 h then 0.1 ml of enrichment broth was streaked on PALCAM agar plates (oxoid CM,877) containing selective supplement (oxoid SR150), then incubated at 30°C for 48 hours. 

Identification of L. Monocytogenes (ISO, 11290):

The suspected colonies (drop-like, black with brown hallow with sunken center) were picked up and subcultured on Trypticase Soya agar supplemented with 0.6%-yeast extract (TSA-YE) and incubated at 30°C for 24then identified according to FAO (1992), Bacterial films from the suspected pure colonies were stained with Gram's stain and using appropriate biochemical tests (Catalase test, H2S production, D-glucose, salacin fermentation (Purple Agar Base, Oxidase test, and Vogas proskouer reaction and umbrella type motility at 22°C. Further identification to species were done using heamolysis on blood agar, Nitrate reduction, acid production from D-xylose, L-raminose, D-Mannitol and methyl-d-mannoside. 

Detection of associated virulence genes of 10 L. monocytogenes isolates by multiplex PCR for :

Bacterial strans : A collection of 10 L. monocytogenes were investigated. 
The collection contained (5,2, 1 and 2) straines from brain, C.S.F., faeces and milk respectivelly from ruminant with listeric encephalitis. 

Multiplex Polymerase chain reaction (PCR) for detection of associated virulence genes of L. monocytogenes:

The multiplex PCR allowed amplification of 9 virulence-associated genes of L. monocytogenes, namely plcA, actA, hlyA and jap, inlB, plcB, inlA, inlC and inlJ. to their respective base pairs, 1484, 839, 456, 131, 146, 261, 800, 517 and 238 bp PCR products, respectively, each represented by a single band in the corresponding region of the DNA marker ladder, all of the amplification products were of the expected size, except for the actA gene did not produced by ay examined strain in this study. 

(

  Chen et al., 2007). L. monocytogenes isolates were small, Grampositive rods, negative for oxidase and urease, and test-positive for methyl red and Voges-Proskauer, had the ability to grow at 35°C, were catalase-positive and motile in wet mounts. They utilized dextrose, esculin and utilized rhaminose with production of acid but failed to utilize xylose Osman et al., (2014(. Clinical manifestations are similar in all susceptible hosts and include septicemia, abortion, severe gastroenteritis, and central nervous system (CNS) infections, such as meningitis, meningoencephalitis, and rhombencephalitis Zhang et al., (2003(. Identification of infected animals was necessary due to the likely causal link to several outbreaks of listeriosis. Rawool et al., (2007). 




  total of 631samples were collected from268 diseased farm animals and emergency slaughtered animals (80 sheep, 60 goat, 75 cattle and 53 buffalo). Such samples including faeces (80), cerebrospinal fluid (17), milk (32) and brain (60) from diseased sheep, faeces (60), cerebrospinal fluid (11), milk (17) and brain(48)] from diseased goat, faeces (75), milk (62) and brain (46) from diseased cattle and faeces (53), milk (41) and brain(29) from diseased buffaloes. These samples were collected from different village of Dakahlia Governorate. Samples were collected separately in sterile plastic bag, well identified and transported in ice box (4 o C) under strict hygienic condition to Mansoura Veterinary laboratory within 2hrs. 




  Photo (1) showed that L. monocytogenes isolates produced (60%), (80%) and (80%) of InlA, InlC and inlJ gene respectively. 




  Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes. Aug; 186(15):4994-5002. -Zhang, C., M. Zhang, J. Ju, J. Nietfeldt, J. Wise, P. M. Terry, Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations. J. Bacteriol. 1855573-5584. ‫مونوسيتوجين‬ ‫الليستيريا‬ ‫ميكروب‬ ‫فى‬ ‫بالضراوة‬ ‫المرتبطة‬ ‫المتعددة‬ ‫الجينات‬ ‫كشف‬ ‫المريضة.‬ ‫المزرعة‬ ‫حيوانات‬ ‫من‬ ‫المعزولة‬ * 




  


Liu et. al., 2007).

  Template DNA extraction according to (AmpliTaq Gold® 360 Master Mix (U.S.A., Applied Bio System Code No.439881) and Oligonucleotides primer 



Table ( 1

 ( ): Oligonucleotides primer sequences and expected product sizes of the multiplex PCR: 


				Expected	
	Gene		Primer sequence (5'3')	product size	Reference
				(bp)	
	inlA	Forward Reverse	ACGAGTAACGGGACAAATGC CCCGACAGTGGTGCTAGATT	800 bp	
	inlC	Forward Reverse	AATTCCCACAGGACACAACC CGGGAATGCAATTTTTCACTA	517 bp	Liu et. al., 2007
	inlJ	Forward Reverse	TGTAACCCCGCTTACACAGTT AGCGGCTTGGCAGTCTAATA	238 bp	
	inlB	Forward Reverse	AAAGCACGATTTCATGGGAG ACATAGCCTTGTTTGGTCGG	146 bp	Sanghun
	plcB	Forward Reverse	GGGAAATTTGACACAGCGTT ATTTTCGGGTAGTCCGCTTT	261bp	et. al., 2012
	plc A	Forward	CTGCTTGAGCGTTCATGTCTCATCCCCC	1484 bp	
					S

. Kaur et. al., 2007

  The mixture used and the reaction conditions for Multiplex PCR for the detection of plcA, hylA, jap and actA were according to S. 


		Reverse	CATGGGTTTCACTCTCCTTCTAC	
	hly A	Forward Reverse	GCAGTTGCAAGCGCTTGGAGTGAA GCAACGTATCCTCCAGAGTGATCG	456 bp
	jap	Forward Reverse	ACAAGCTGCACCTGTTGCAG TGACAGCGTGTGTAGTAGCA	131bp
	act A	Forward Reverse	CGCCGCGGAAATTAAAAAAAGA ACGAAGGAACCGGGCTGCTAG	839bp
	The mixture used and the reaction conditions for Multiplex PCR for
	the detection of inlB, plcB were according to Sanghun et. al., (2012).

The mixture used and the reaction conditions for Multiplex PCR for the detection of inlA, inlC and inlJ were according to Liu et.al.,(2007). 
Kaur et. al., (2007) Agarose gel electrophoresis was done according to Sambrook et al., (1989). RESULTS Total incidence of Listeria monocytogenes in diseased farm animals samples by culture isolation method: It

  
		Type and number of samples			Positive		
	Animals	No. of animals	Faeces	Milk	Brain	C.S.F.	Total

is evident from the table (2) that the prevalence of L. monocytogenes in diseased sheep was (36.5) the higher rate of isolation was from C.S.F (47.05%) followed by brain (43.3%), milk (32.5%) and from feaces (28.1%).The prevalence of L. monocytogenes in diseased goat was (28.67) isolated from (23.3%), (29.4%), (31.25%) and (36.36%) from feaces, milk, brain, and C.S.F respectively, while in diseased cattle the prevalence of L. monocytogenes was (24.48) from faeces (28%), milk (20.9%) and brain (24.48%).As shown in table (2) that the prevalence of L. monocytogenes in diseased buffaloes isolated in a ratio of (26.4%), (12.1), and (10.3%) from faeces, milk and brain respectively.Table (2):Total incidence of L. monocytogenes in diseased farm animals samples by culture isolation method: 
Detection Of Virulence Associated Genes In ...

  
												Ahmed Ali El Sawaak. et., al.
	Sheep	80	80	32	60	17	189	26	32.5	9	28.1	26	43.3	8	47.05	69	36.5
	Goat	60	60	17	48	11	136	14	23.3	5	29.4	15	31.25	4	36.36	39	28.67
	Cattle	75	75	62	46	0	183	21	28	13	20.9	13	24.48	0	0	48	24.48
	Buffaloe	53	53	41	29	0	123	14	26.4	5	12.1	3	10.3	0	0	24	17.4
	Total	268	268	152	183	28	631	75	27.98	37	20.55	47	25.68	12	42.85	180	27.31
																	303

Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 
Table ( 3): Detection

 ( of virulence associated genes in 10 L. monocytogenes 


	isolates by multiplex PCR:

No. of the isolates Virulance associated genes Animal species

  
	10	+	+	-	-	+	+	-	+	+	buffloe	Brain
												Origin of
		inlA	inlC	inlJ	inlB	,plcB plcA	actA	hlyA iap		the sample
	1	-	-	-	-	-	-	-	-	-	Sheep	Feaces
	2	-	-	-	-	-	-	-	+	+	cattle	Milk
	3	+	+	+	-	+	-	-	+	+	Sheep	C.S.F
	4	-	+	+	-	+	-	-	+	+	cattle	Brain
	5	-	+	+	-	-	-	-	+	+	Sheep	Milk
	6	+	+	+	+	+	+	-	+	+	Sheep	Brain
	7	+	+	+	+	+	+	-	+	+	Sheep	C.S.F
	8	+	+	+	+	+	+	-	+	+	goat	Brain
	9	+	+	+	-	+	+	-	+	+	cattle	Brain

Detection Of Virulence Associated Genes In ...

  
	Ahmed Ali El Sawaak. et., al.
	from the examined samples +ve: positive control of L.
	monocytogenes and -ve: negative.
	DISCUSSION
	Enrichment of L. monocytogenes was done on the UVMI and UVMII
	broth (Jemmi and Keusch 1994). Isolation of L. monocytogenes were
	done on the PALCAM media with selective supplement, typical colonies
	recovered were grey-green 1.5-5 in diameter, and have black sunken
	centers due to esculin hydrolysis as the results obtained by (Parihar et.
	al., 2008).
	Results in table (2) for the bacteriological examination on 268
	diseased farm animals and emergency slaughtered animals (80 sheep, 60
	goat, 75 cattle and 53 buffalo) revealed that (36.5%, 28.6%, 24.48% and
	17.4 %) isolated from sheep, goat, cattle and buffaloes respectively were
	nearly similar to the results that recorded by (
	306

Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 
Simranpreet et. al., (2010); Antônio et. al., 2014; Osman et al., 2014). Higher figures were reported by Esteban et. al., (2009).

  Meanwhile lower prevalence was recorded byThe isolation of L. monocytogenes from fecal samples of diseased cattle agrees with F. 



A. Lawan et. al., (2013).

  
	Isolation of L. monocytogenes from milk samples of diseased cattle
	agrees with Alejandra et. al., (2009); while higher rate of isolation of L.
	monocytogenes recorded by Deepansh et. al., (2012).
	Table (2) approved that L. monocytogenes recovery rate from the
	brain, feaces and milk of the examined buffaloes were (10.3 %), (26.4%),

(12.1%) respectively, these results agree with Simranpreet et.al., (2010).Lower rate of isolation was recorded by Chaudharia et.al., (2004).The isolation of L. monocytogenes from milk samples of diseased buffloes agrees with Deepansh 
et. al., (2012); Farad et. al., (2013).

  
	Multiplex PCR assay was carried out for detection of L.
	monocytogenes and presence of inLA, inLC, inlJ, inlB, plcB, plcA, hlyA,
	actA and jap genes.
	However, some L. monocytogenes strain may lack one or more
	virulence determinants because of some mutation

Cooray et. al., (1994).

  
	produced inlB gene. InlB is not an important virulence factor for buffaloes Comparative Immunology, Microbiology and Infectious
	listeriosis Liu et. al., (2007) recognized that InlB gene failed to be Diseases Volume 27, Issue 2, March 2004, Pages 141-148.
	recognized with serotype 4 strains. -Chen, Y., W. Zhang, and S. J. Knabel. (2007): Multi-virulence-locus
	The result in photo (3) that (90%) of L. monocytogenes isolates sequence typing identifies single nucleotide polymorphisms which
	produced hylA gene, (80%) of L. monocytogenes isolates produced jap differentiate epidemic clones and outbreak strains of Listeria
	gene and (50%) of L. monocytogenes isolates produced plcA gene. monocytogenes. J. Clin. Microbiol. 45835-846)
	Roche et. al., (2009) stated that some isolates of L. monocytogenes -Cooray, K. J., Nishibori, T., Xiong, H., Matsuyama, T., Fujita, M.
	harbor inlA gene with or without hlyA gene which supported the and Mitsuyama, M. (1994): Detection of multiple virulence-
	usefulness of studying the pathogenic potential of strains. associated genes of Listeria monocytogenes by PCR in artificially
	contaminated milk samples. Appl. Environ. Microbiol. 60:3023-3026.
	As shown in photo (1) L. monocytogenes isolates produced (60%),
	(80%) and (80%) of InlA, InlC and inlJ gene respectively. The combined
	application of inLA which is species-specific, inLC and inLJ gene
	primers in a multiplex PCR confirm L. monocytogenes species identity
	and its potential virulence Jaradat et. al., (2002).
	The results in photo (2) show that (70%) of L. monocytogenes
	isolates produced plcB gene and (30%) of L. monocytogenes isolates

-Deepansh Sharma; Pradeep Kumar Sharma; B. S. Saharan2 and Anjali Malik (2012): International Journal of Microbial Resource Technology, Vol.1, No.1 (Jan 2012), 1-4 1.-Ebrahim 
Rahimi, Hassan Momtaz, Asma Behzadnia and Zeinab Torki Baghbadorani (2014):

  Incidence of Listeria species in bovine, 


	ovine, caprine, camel and water buffalo milk using cultural method
	and the PCR assay Asian Pac J Trop Dis. 2014 Feb; 4(1): 50-53.
	-Elgamal A.M. (2003): Bacteriological Investigation of Listeriosis
	Epizootic in Sheep Flock in El-Dakhlia province. Zag. Vet. J. (ISSN.
	1110-1458) Vol 31, No., (2003) PP. 85-90.
	-Esteban, J.I.; Oporto, B.; Aduriz, G.; Juste,R.A.; and Hurtado,
	A.; (2009): Faecal shedding and strain diversity of Listeria

F. A. Lawan et. al., (2013).

The variation in the isolation rate may be due to variation in methods of isolation Jemmi and Keusch, (1994) and variation in animal husbandry practice and type of animal species F. A. Lawan et. al., (2013). 

Results in

Table (2) showed that L. monocytogenes was recovered from the C.S.F., brain, faeces and milk of the examined sheep was (47.05%), (43.3 %), (32.5%), and (28.1%) respectively. These results agree with (Elgamal A. M. 2003; Antônio et. al., 2014). 
Detection Of Virulence Associated Genes In ... Ahmed Ali El Sawaak. et., al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 307 The results achieved of the isolation of L. monocytogenes from fecal samples of sheep agree with F. A. Lawan et al., (2013). Lower rate of isolation were recognized by Esteban et. al., (2009). The isolation of L. monocytogenes from sheep milk samples agree with Osman et al., (2014). Lower rate of prevalence recorded by Ebrahim et. al., (2014). Table (2) declares that that L. monocytogenes was recovered (36.36%), (31.25 %), (23.3%) and (29.4%) from the C.S.F., brain, feaces and milk of the examined goats. The isolation rates of L. monocytogenes from C.S.F. and brain samples of diseased goat were in the ranges which agree with Antônio et. al., (2014). Lower rate of isolation were recognized by Shivasharanappa et. al., (2014). 
The result of isolation of L. monocytogenes from milk of diseased goat samples agrees with Osman et. al., (2014). The lower rate recognized by Ebrahim et. al., (2014). The isolation of L. monocytogenes from faeces of diseased goat samples were in the range which agrees with F. A. Lawan et. al., (2013); Farad et. al., (2013) L. monocytogenes were recovered from the brain, faeces and milk of the examined cattle in a ratio of (24.48%), (28%) and (20.9%) respectively as shown in Table (2); which agree with Antônio et. al., monocytogenes in healthy ruminants and swine in Northern Spain BMC Veterinary Research 2009, 5:2 . -FAO (1992): Manual of food quality control. 4 Rev. Chapter 11, pp. 119-129. 1. Microbiological analysis of food and Agriculture Organization of the United Nations, Rome. -Farad Safarpoor Dehkordi; Sara Barati; Hassan Momtaz; Seyyedeh Nasib Hosseini Ahari; and Shahin Nejat Dehkordi (2013): Comparison of Shedding, and Antibiotic Resistance Properties of Listeria monocytogenes Isolated From Milk, Feces, Urine, and Vaginal Secretion of Bovine, Ovine, Caprine, Buffalo, and Camel Species in Iran. Journal of Microbiology. 2013 May; 6(3): 284-94. -FSIS ˝United State Department of Agriculture, Food Safety Inspection Service (1989): Method for the isolation and identification of Listeria monocytogenes from meat and poultry products. Laboratory Communities. No. 57, US Department of Agriculture, Washington, D.C. -F. A. Lawan; A. N. Tijjani; A. I. Raufu; J. A. Ameh; I. Y. Ngoshe and M. S. Auwal (2013): Isolation and characterisation of Listeria species from ruminants in Maiduguri north-eastern Nigeria. African Journal of Biotechnology, Vol. 12(50), pp. 6997-7001. -Jaradat ZW; Schutze GE ;Bhunia AK (2002): Genetic homogeneity among Listeria monocytogenesstrains from infected patients and meat products from two geographic locations determined by phenotyping,
Ahmed Ali, El Sawaak, Ibrahim El Sayed, El Desoky, Ahmed Mohamed, Abd Elgwaad, Hanaa A Ahmed, Marwa Ibrahim, El Morsy
Detection Of Virulence Associated Genes