SECOND WORKSHOP OF NATIONAL REFERENCE LABORATORIES FOR

PARASITES14-15 JUNE, 2007

ISTITUTO SUPERIORE DI SANITURoom Daniel Bovet, Viale Regina Elena, 299

00161 Rome Italy

Dr Andreas PhylactouVeterinarian

CYPRUS

Information

• The third-largest island in the Mediterranean Cyprus lies off the southern coast of Turkey and the western shore of Syria. The highest peak is Mount Olympus at 6,406 ft (1,953 m).

• Land area: 3,568 sq mi (9,241 sq km);

• Total area: 3,571 sq mi • Population (2006 est.): 784,301

(growth rate: 0.5%); life expectancy: 77.8; density per sq mi: 220

Veterinary Services of Cyprus

Veterinary Services Organogram

DIRECTOR

REGISTRY ACCOUNTS

STORE

EUROPEAN UNION AND

INTERNATIONAL ORGANIZATIONS SECTION INTERNAL AUDITING SECTION

ANIMAL HEALTH AND WELFARE

DIVISION

VETERINARY PUBLIC HEATLH

DIVISION

VETERINARY LABORATORIES SECTION

DRUGS AND SUPPLIES SECTION

D.V.O.

NICOSIA

D.V.O. LIMASSOL

D.V.O. LARNACA

D.V.O. AMMOCHOSTOS

D.V.O. PAPHOS

R.V.S. AVDEMOU

R.V.S. SKARINOU

R.V.S. P. CHRYSOCHOUS

R.V.S. NISOU R.V.S. AKAKI

Laboratory for the Control of Food of Animal Origin

VETERINARY LABORATORIES

QUALITY MANAGERDEPUTY SAFETY MANAGER

A. PHYLACTOU

SAFETY MANAGERDEPUTY QUALITY MANAGER

G. CHRISTOFI

HEAD OF VETERINARYLABORATORIES

KYRIAKOS GERGIOU

VIROLOGY

DIRECTOR OF VETERINARY SERVICES GIORGOS NEOPHYTOU

SCIENTIFIC STAFFK. GEORGIOU

(TECH. MANAGER)

P. STYLIANOUA.MAZERIS

(QUALITY MANAGER)

TECHNICAL STAFF

A. SHIAKALLISS. PAPANEOPH.M. NIKOLAOUM HATJIYANNI

THEOPHANOUSS. MINA

D. THEMISTOKL.

SCIENTIFIC STAFFI. IOANNOU)

TECHNICAL STAFF

N. CHRISTOFIC. ZINIERIS

SCIENTIFIC STAFFI. IOANNOU

(TECH. MANAGER)

TECHNICAL STAFFM. SAVVIDES

Z.POLYCARPOUT. DEMETRIOUS. DAMIANOU

SCIENTIFIC STAFFSOTIRIA GEORGIADOU

(TECH. MANAGER)

TECHNICAL STAFF

N. THEOPHILOU

SCIENTIFIC STAFFM. LIAPI

(TECH. MANAGER)

TECHNICAL STAFFM. AVRAAME. TSINTIDOUM. ANDREOUP. IOANNOU

N .PAPAEFSTATHIOU

LCFAOBACTERIOLOGY/

SEROLOGY HISTOPATHOLOGY

POST MORTEM-MICROBIOLOGY

PARASITOLOGY

TECHNICAL STAFFK. ACHILLEOS

F. TOUMBAM. SHIAKALLISA.TOFALLIDOU

S. CONSTANTINOUA. DIMOSTHENOUS

M. OMIROUAFROYLA SKOTTI

SCIENTIFIC STAFFANDREAS PHYLACTOU

C. ECONOMIDESMARIA EMMANUEL

CLEANERSA. NIKOLAOUK. KYRIACOU

A. PAVLOUC. MONOGIOU

BIOLOGICALPRODUCTS

VETERINARY LABORATORIES

Veterinary Services of Cyprus • Laboratory for the Control of Foods of Animal Origin

(LCFAO) is the Cyprus National Reference Laboratory for Trichinella spiralis

• Dr Kyriakos GeorgiouHead of Veterinary Laboratories• Dr Andreas PhylactouHead of LCFAO• Dr Ioannis IoannouOfficer in Charge for the Laboratory of Parasites

Mission of Veterinary Services

• The Cyprus Veterinary Services are the Competent Authority for the control of foods of animal origin.

• LCFAO provides services through microbiological, parasitological, presence of residues and physicochemical examinations on foods of animal origin including water.

• Control of exported and imported foods of animal origin for their suitability.

• LCFAO• Is based in Nicosia in the same block with the Headquarters of the

Veterinary Services.• Is responsible for the control of foods of animal origin for the whole

island.

PersonnelHead of LCFAO (Vet. Off.)Quality Manager (Vet. Off.)

Technical Manager (Vet. Off.)Technical Manager Assistant (Vet. Off.)

8 Laboratory Technicians1 Secretariat

1 Glassware cleaning & Sterilization1 Cleaning Staff

Structure of LCFAO• Sample Registration, Management and Dispatching

Unit

• Media Preparation Unit

• Raw Milk Unit• Total bacterial count at 30 οC (ISO 6610/1992)• Somatic cells (ISO 13366-1, Microscopic method)

(ISO 13366-3, Fluoro-opto-electronic method)• Freezing point (Dir. 91/180 EEC)• Presence of antibiotics (DELVO-Test)

Pasteurized Milk and Milk Products Unit• Total count at 30 οC (ISO 6610/1992)• Staphylococcus aureus (ISO 6888-1/1999)• Clostridium perfringens (Iron sulfit agar)• Bacillus cereus (ISO 7932/1998)• Coliforms (ISO 5541-1/1986)• Escherichia coli (ISO 16649-2/2001)• Yeast and Molds (ISO 7954/1987)• Psychrotrophic microorganisms (IDF 132A)• Phosphatase (Lactognost®)• Peroxydase (Dir. 91/180 EEC)

• Meat and Meat Products, Fish, Eggs, Water Unit• Total bacterial count at 30 οC (ISO 6610/1992)• Staphylococcus aureus (ISO 6888-2/1999)• Clostridium perfringens (Iron sulfit agar)• Coliforms (Meat: ISO 4832/2001)

Water: ISO 9308-2MPN/1990)• Escherichia coli 0157(Meat:16654/2001

Water: ISO 9308-2 MPN/1990)• Trichinella spiralis (Trichinoscope, Digestion

method, Commission regulation 2075/2005/EC)

• Food borne Pathogens, Mastitis Unit• Salmonella spp. (ISO 6579/2002)• Salmonella Susceptibility testing (NCCLS

Guidelines)• Listeria (ISO 11290-1/1996, 11290-2/1998)• Campylobacter (direct plating on Camby-agar)• E. coli 0157 (ISO 16654/2001)• Mastitis (antibiogramme)

• Veterinary Drug Residues and Staphylococcal Enterotoxin Unit:

• FPT (Dir. 86/469/EEC,96/23/EEC)• Sulfonamides (ELISA, TLC)• Staphylococcal Enterotoxin (Transia -

Reference method of the CRL, V.2, September 2005)

• Histamine detection (TLC)

Monitoring Programs of LCFAO

• Somatic Cell Count in raw milk• Total bacterial count of raw milk• Antibiotics in raw milk• Veterinary drug residues in meat• Salmonella, Listeria, Campylobacter in

poultry carcasses• Trichinella spiralis

Accreditation - ISO 17025/2005

8 July 2003 accredited for ISO 17025/1999by the Greek Accreditation Body ESYD• Total Bacterial Count in Raw and Pasteurized milk.• Somatic Cell Count-Microscopic method• Somatic Cell Count-Fluoro-opto-electronic method• Listeria Detection• Salmonella Detection• Four Plate Test (FPT)• Staphylococcal Enterotoxin

Trichinella spiralis

• CLASS: ADENOPHOREA • SUBCLASS: ENOPLIA • ORDER: STICHOSOMIDA

(TRICHURATA) • SUPERFAMILY:

TRICHOCEPHALOIDEA • FAMILY: TRICHINELLIDAE

Scientific name - Trichinella spiralis

• Disease - trichinosis

Life Cycle

• This nematode has no stages outside a host. The adult worms are found attached to or buried in the mucosa of the duodenum. Aftermating the males die. Females (ovoviviparous) produce living young (approximately 1,500 per female over a period of 4 to 16 weeks) and then die. Juveniles enter the lymphatics and mesenteric veins and are found throughout the arterial circulation between the 7th and 25th day after infection. They travel in the hepatoportal system through the liver, then to the heart, lungs, and the arterial system, which distributes them throughout the body. They are transported to striated muscles, penetrate individual fibers, and cysts are formed around the juveniles. Within cysts, juveniles remain viable for many years, up to 25 years in man and 11 years in pigs. There is agreement that there are four juvenile stages but disagreement in the literature as to whether or not nematode development occurs within the cyst. When viable encysted juveniles are ingested, they are digested from the cysts and pass to the duodenum where they mature. Humans are essentially a dead end for the parasites.

Symptoms-Pathogenicity

• Nausea, dysentery, colic due to invasion by adult worms. • Migrating juveniles cause pain as they invade muscle

tissue; there may also be edema (swelling), delirium, cardiac and pulmonary difficulty, pneumonia, nervous disorders, deafness and delayed or lost reflexes.

• Many cases are never diagnosed because of the vaugeness of the symptoms. Muscle biopsy can be conducted and involves pressing muscle between glass plates to look for cysts. Xenodiagnosis involves feeding suspected muscle to laboratory rats.

Management - Prevention

• Cook pork thoroughly (also flesh of bear, walrus, wild pigs). • Cook all garbage fed to hogs. • Proper meat handling, ordinary curing and salting of pork products will not kill

encysted juveniles. • Freezing is effective if carried out properly. The freezing requirements differ with the

size of the meat. Pieces not exceeding 6 inches in thickness require 20 days at 5F, 10 days at -10F, 6 days at -20F. Larger pieces require longer periods. Quick freezing and storage for 2 days is effective.

• Although there have been marked reductions in the percentage of hogs carrying encysted juveniles in the United States, there is considerable variation from one part of the United States to another. It is therefore a wise precaution to avoid uncooked pork products. In the United States Trichinella occurred in 0.4 to 2.5% of the prepared pork products examined in some areas (1961); however some areas had as much as 10% infection. In Chile 0.22% of 500,000 hogs were infected (1964). In Germany only 0.00026% of 14,000,000 hogs were infected (1965). It has been estimated that 3/4 of the persons infected with Trichinella are from North America.

• Treatment of Host: No cure. Relieve symptoms with analgesics and corticosteroids.

Characteristics

• The males measure 1.4 to 1.6 mm long and are more slender at the anterior than the posterior end. The anus is nearly terminal and has a large papilla on each side of it. A copulatory spicule is absent. Stichocytes are arranged in a row following a short muscular esophagus.

• Females are about twice the size of males, also tapering toward the anterior end. The anus is nearly terminal. The vulva is located near the middle of the esophagus, which is about a third the length of the body. The single uterus is filled with developing eggs in its posterior portion, whereas the anterior portion contains fully developed, hatching juveniles.

• Adult female male infective muscle larvae

Historical Data of Trichinella in Cyprus

• Trichinellosis is a notifiable parasitic disease in Cyprus. • However it has never been observed in the island. • During 1969 and annually afterwards, pig carcasses were periodically

tested for the presence of Τrichinella spiralis from slaughterhouses all over Cyprus.

• Since 1999 a more systematic surveillance program for Trichinella testing has been established. Samples were collected from the pillarsof the diaphragm of pig carcasses including sows and boars, from all the pig farms all over the island. The last seven years (1999-2005) animals from the wild life (foxes, stray dogs and cats, rodents) werealso included.

• From 1969 until January 2004, the Trichinella spiralis testing was performed centrally in the Laboratory for the Control of Foods of Animal Origin of the Cyprus Veterinary Services.

• Originally meat samples were examined with the Trichinoscope whilethe last 10 years the method of choice is the digestion method with the use of Pepsin and Hydrochloric acid.

• Since January 2004, the examination of fresh meat derived of domestic swine is performed at the slaughterhouses laboratories.

• During 2004 a number of 357638 swine meat samples were examined. These animals originated from all 121 piggeries of Cyprus and covered80% of the target population.

• During 2005 and 2006 all pigs slaughtered in Cyprus were tested for Trichinella.

• The testing of samples from the wild life and from other domestic animals is performed only at the LCFA. (2006-108 samples)

• There are no piggeries in the non controlled by the Government of Cyprus area. The reason for this is that Muslim population (Turkish Cypriots, Settlers and soldiers from Turkey) living in this part of the island, are not consuming pork meat of religious reasons. They are Muslims.

• There are no wild pigs in Cyprus and the fox population is very low.

Wild life

• During the last seven years 1288 samples from the wild life were tested.

• All samples were found negative for Trichinella.• The last few years a great number of stray dogs and foxes were

examined for Trichinella, after were captured and killed by United Nations in the buffer zone, separating the controlled and the non controlled area by the Government of Cyprus, based on the antiechinococcosis campaign programme supported by UN (UNOPS).

• None of these animals was found positive for Trichinella.• The same was the case with the testing of rats for Trichinella where

many rats were also captured from areas close to the buffer zone.

Wild life

1288355104052487210692Total

1----1--Goats

8---62--Hares

12--13332Mouflons

1-----1-Horses

361235-718Foxes

938354-386196-11Rats

4----2-2Mice

5---41--Stray cats

283-81534639469Stray dogs

Total

2005 (-May)200420032002200120001999Animal

species

Trichinella testing in Cyprus after 1974

NIL19241994

NIL41741993

NIL3181992

NIL2471991

NIL101990

NIL4721989

NIL2141988

NIL1071987

NIL1881986

NIL4241985

NIL7221984

NIL1341983

Positive samplesNo of samples examinedYear

Trichinella testing in Cyprus after 1974

NIL6477292006

NIL6658782005

NIL3576432004

NIL23992003

NIL12902002

NIL13392001

NIL14122000

NIL8281999

NIL4171998

NIL3021997

NIL1811996

NIL1871995

Positive samplesNo of samples examinedYear

Trichinella spiralis has never existed in CyprusSurveillance program is in place according to Directive 77/96

4209

386 (weasels)

3

15

3805

2003

-208 (weasels)

910Other Animals

2004200220012000

255292

131113421412Total

3537Foxes

8376394Dogs

255281

106112671301Swine

4209

386 (weasels)

3

15

3805

2003

-208 (weasels)

910Other Animals

2004200220012000

255292131113421412Total

3537Foxes

8376394Dogs

255281106112671301Swine

Toxoplasmosis

• Serological examinations (indirect haemagglutination test) in sheep and goats are showing positive results and the disease is considered to be one cause of abortions.

• In humans although there are no official reports for the disease, it is considered to be a problem in the female population.

• The problem needs further investigation.

Cryptosporidiosis

• Only feaces sample from diarrheic calves, lambs and kids examined

Method usedRapid testModified Zichl Nielsen coloring

Cryptosporidiosis

124

3121Total

3382006

242005

592004

CalvesLamb and kidsYear

E. Echinococcosis

• 1971-1985: Eradication program total eradication

• 1994: New eradication program

Surveillance program in place monitors:

• Registration and identification of all dogs with microchip (obligatory) • Dog treatment:

– preventive treatment with praziquantel (required also for imported dogs)

• Stray dog control – praziquantel baits to the buffer zone

• Dog testing: – faeces samples tested with Coproantigen ELISA test

Surveillance program in place monitors:

• Slaughtering inspections: – inspection of all kinds of carcasses

• Dead animals:– Proper disposal of carcasses

• Public enlightening: – lectures at clubs, schools, army camps etc.

Echinococcus

ANIMALS (cows, sheep & goats) INFECTED WITH CYSTS

13

19

13

2

9

1

8

10

0

2

4

6

8

10

12

14

16

18

20

1997 1998 1999 2000 2001 2002 2003 2004 2005YEAR

NO

. OF

ANIM

ALS

NO. OF ANIMALS INFECTED

Farm animals found infected in slaughterhouse inspectionsDogs: Since 1995 no infected dog has been found

00000≈ 2500002005

100109812602004

8023310098952003

100109781862002

900909392822001

201109274322000

13011028994461999

1915948962261998

1310938954711997

TOTALMoufflonsGoatsSheepBovine

INFECTED ANIMALSAnimals slaughtered

Year

Μόλυνση µε Κύστεις Εχινόκοκκου –Αίγες

____14,362____130,5651985

0,0119,348____103,4141984

0,01110,084____99,5701983

0,01110,220____100,4371982

0,03412,391____101,8381981

0,131612,715____117,0121980

0,224118,250____119,7121979

0,326620,570____131,8871978

0,679013,476____111,6091977

1,831699,243____101,5381976

3,913178,1010,007689,4571975

4,932655,3790,0181583,6201974

8,841,45116,4050,0896122,9341973

12,511,32110,5560,966575,6251972

15,013682,4511,460041,5331971

ΠοσοστόΜόλυνσης

Μολυσµένα ΖώαΑίγες άνω των 2 ετών

ΠοσοστάΜόλυνσης

Μολυσµένα ΖώαΑίγες κάτω των 2 ετών

Έτος

Μόλυνση µε Κύστεις Εχινόκοκκου – Πρόβατα

0,112421,779__198,1231985

0,498216,576__158,1321984

0,7913416,884__154,7491983

1,0819017,512__164,4381982

3,0457018,754__158,1001981

4,1578018,809__176,7311980

7,271,67723,080__180,2581979

12,763,09724,262__187,3971978

14,862,63717,746__140,7071977

24,093,20013,2830,00091113,7321976

30,443,77812,4100,0252391,4461975

43,413,4968,0530,0898124,4551974

10,731,70215,8610,21304146,7321973

53,856,17511,4661,291,21893,9921972

49,631,8193,6652,281,36859,8761971

ΠοσοστόΜόλυνσης

Μολυσµένα ΖώαΠρόβατα άνω των 2 ετών

ΠοσοστόΜόλυνσης

ΜολυσµέναΖώα

Πρόβατα κάτω των2 ετών

Έτος

Μόλυνση µε Κύστεις Εχινόκοκκου – Βοοειδή

0,0412,321____6,0131985

0,6291,457____6,0401984

1,38191,376____6,3431983

3,41401,174____5,0641982

5,85871,487____5,2201981

2,63351,333____5,5471980

5,42721,329____5,1301979

5,65921,627____5,8501978

4,08591,446____4,9181977

8,051151,4290,0734,5001976

10,201541,5120,0734,1211975

21,03671,7430,2094,4261974

35,101,1593,3060,26228,4051973

30,68612,8120,9485,0741972

38,901,0322,6521,8915,2751971

ΠοσοστόΜόλυνσης

Μολυσµένα ΖώαΒοοειδή άνω των 2 ετών

ΠοσοστόΜόλυνσης

Μολυσµένα ΖώαΒοοειδή κάτωτων 2 ετών

Έτος

Μόλυνση µε Κύστεις Εχινόκοκκου – Χοίροι

____5,565____294,2171985

____5,269____298,8861984

____4,431____269,1431983

____3,514____246,9241982

____2,691____221,4021981

____3,695____181,9441980

____4,164____195,9501979

____6,281____186,1251978

0,0425,584____157,8971977

0,2751,8500,0012136,5641976

4,71282,7200,0023123,4971975

10,23803,7340,02738142,8221974

7,44365,8840,0799134,9061973

10,04404,3980,2219788,7511972

18,607644,1160,8257570,0011971

ΠοσοστόΜόλυνσης

Μολυσµένα ΖώαΧοίροι άνω των 2 ετών

ΠοσοστόΜόλυνσης

ΜολυσµέναΖώα

Χοίροι κάτω των 2 ετών

Έτος

Αριθµός δολωµάτων Praziquantel πουχρησιµοποιήθηκαν

88741163711241Σύνολο

100023501367Πάφος

176611641400Αµµόχωστος

30101188976Λεµεσός

180036435248Λάρνακα

129632922250Λευκωσία

200620052004Επαρχία

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