EUFMD- ETC 5 FMD REAL TIME TRAINING COURSE ERZURUM 03-06 MAY 2010

FMD CLINICAL AND EPIDEMIOLOGIAL REPORT: ESENKENT 04/05/10

Introduction

Suspicion of Foot and Mouth Disease (FMD) was reported in the village of Esenkent on Monday 3rd May 2010. The official veterinarian inspected the herd, to find clinical signs suggestive of FMD. Samples at this time were taken to support the diagnosis of FMD based on the the clinical findings, and were sent to the SAP institute.

The village was visited by the ETC-5 team on Tuesday 4th May. The EUFMD-training team

was split into two groups to carry out further clinical examination, sampling, and to

undertake an epidemiological investigation.

The location

Esenkent is a small village located 20km from the Armenian border. Esenkent is located in

the Kars district of the Turkish province of Kars. The village is located 40 km away from the

capital of the province, Kars city. The human population of the village is 200. The animal

barns, which house the animals in winter, are interspersed throughout the village,

distributed between the many residential buildings in which the villagers live. There are

common grazing grounds around the village, where all animals (irrespective of owner) graze

at certain times in the year. A satellite image of the village, along with locations of the 5

barns which contained the animals examined, is shown in Fig.1.

Population at risk

Data given by the local farmers and the local official veterinarian, gave animal population

sizes in the village of 1160 cattle and 100 sheep for fattening. There are no goats and no

pigs. At this time of year, animals are all out to graze on the common pastures, with only

animals which are sick being housed in the many barns in the village.

These animals are subdivided by ownership, with 60 different farmers being present in the

village, thus the term “farm” is used to described those animals belonging to one farmer.

FMD history

The last FMD outbreak in Esenkent was reported to be in 2006. FMD is endemic in the

Anatolia region of turkey.

FMD serotype ‘O’ has been reported recently in surrounding villages. At this point we are

unable to quantify, date or pinpoint these.

Upon arrival of the local veterinarian, it was reported by the villagers that the clinical signs

which lead to the local vet being called out first occurred on the 28th April. These clinical

signs were reported to be consistent with FMD and included pyrexia, vesicular lesions and

erosions on the lips and tongue, and general lethargy. As stated, the date of notification was

3rd May.

It was reported that, on May 3rd (the day before the ETC-5 team arrived), 20 animals were

showing clinical signs consistent with FMDV infection. These were all cattle, and belonged to

5 farms.

Vaccination regime

Vaccination is compulsory in Kars, and is financed by the local administration. Cattle must be

vaccinated, with a trivalent vaccine, twice a year and sheep once a year. The first

vaccination being carried out at 2 months of age and a second booster being given 1 month

later. Vaccination must be recorded in the passport and on the Turkish database, ‘TurkVet’.

The local veterinarian alone is permitted to vaccinate the animals.

Movement of animals. Source and destination.

Movement out of the village is only allowed for vaccinated animals and they should to be

accompanied by a health certificate.

It is a common practice to buy and sell animals in the local livestock market in Kars city.

Identification and registration of animals

Tagging and registration is official and compulsory within 6 months after birth with the

application of two ear tags and the issuing of an individual passport. In small ruminants the

system is under development but in cattle this is mandatory.

Fig.1. A satellite image of Esenkent, showing the structure of the village and also the locations of the 5 Barnes visited as part of the ETC-5 visit. The entrance of the village acted as our biosecurity and disinfeciton checkpoint and is also indicated.

Epidemiological evaluation of risk factors

Herd management

During winter, cattle are housed inside the barns. Depending on the local climate and

pasture quality / growth, the animals are released from the barns after winter, to graze on

pastures. These pastures are grazed by all animals in the village, irrespective of owner

(farmer). In spring and summer, for a period of 3 to 4 months animals are moved to

common upland pastures (yayla). These are common to many villages.

During winter, the animals are separated spacially as they are housed inside barns. However

these barns are in close proximity to each other, and upon questioning and observation,

there do not appear to be any biosecurity measures in place.

Outside the winter months, as outlined above, all animals from the 60 different farms are all

grazed together.

In addition, it is practiced to allow sick animals to graze in the common pastures, as this is

the only source of feed that they have. As all animals in the village use common grazing

ground, this is a major risk factor for the spread of FMDV

These facts, no clear farm separations, normal lack of biosecutiry measures and intermixing

of animals, lead us to group the whole village as one epidemiological unit with respect to

FMD epidemiological investigation.

Identification and registration

In the village (and on the Erzurum livestock market (visited on 5th May), it was observed

that the majority of the cattle were correctly identified via ear tag. There were, however, a

proportion of animals that were not identified via ear tag. This makes subsequent back and

forward tracing much more difficult, as well as vaccination recording and registration of

animals on the ‘TurkVet’ database.

Animal movement

Back Tracing

Upon epidemiological questioning of the local farmers, it was discovered that one month

ago, 20 cattle were bought at the Kars livestock market and brought to the village. Further

questioning revealed that some of these animals had showed clinical signs suggestive of

FMD, but were not reported at the time. It was reported that these animals have now

recovered and were out grazing with the rest of the village animals at the time of the ETC-5

visit and therefore not available for clinical examination / sampling by the ETC-5 team.

Tracing forward

It must be highlighted that 30 animals were sold 3 weeks ago to a dealer, who had the

intention to transport the animals to Konya (1.177 Km westwards).

Nevertheless, prior to this movement the official veterinarian inspected the animals,

checking correct vaccination, absence of clinical signs and passports.

Vaccination

The official veterinarian informed the team that on 3 March, 1078 animals were vaccinated.

There is a difference between the number of animals vaccinated and the number of animals

present in the village. The reason for this, according to the official veterinarian, is that only

healthy animals are vaccinated. As well, gravid animals are not vaccinated.

After the inspection it was noted that following the check of the vaccination records of 6

animals in the database, 3 animals were recorded to be vaccinated for the last time in

Autumn 2009 and 3 have not any record of vaccination. This may be a problem of incorrect

vaccination, or of incorrect introduction of the data in the database.

Epidemiological Overview details of animals examined by ETC-5

Upon arrival at Esenkent village, most of the village animals were out at pasture. It was

explained that those which had been seen to be showing signs consistent with FMD had

been moved inside the barns in the village by the respective farmers. The animals in each

barn were thus part of a larger farm, and were all owned by a single farmer:

First set of infected animals examined (Barn 1)

Three animals were in this barn at the time of inspection. The others belonging to this

farmer were grazing outside. All of this farmer’s animals were born at the farm.

Second set of infected animals examined (Barn 2)

Two animals were in this barn at the time of inspection. This farmer owned 20 animals, 14

of which had shown clinical signs but 12 are already recovered. This farmer had bought 9

cattle from the Kars market within the last month. It was not reported that these animals

had shown signs suggestive of FMD and these animals were not available for examination.

Third set of infected animals examined (Barn 3)

About 20 young cattle were present in this barn at the time of inspection. Although all

young, the age range amongst these animals appeared large. It was suggested that these

animals were housed due to their young age. The farmer was unwilling for extensive

examination and handling of all animals in this barn, but upon observation, one cow was

seen to be showing signs suggestive of FMD and thus was permitted to be subject to

thorough examination by the ETC-5 team. lThis farmer owned 90 cttle in total, the rest

being turned out to graze on the common pasture.

Fourth set of infected animals examined (Barn 4)

Two animals were in this barn at the time of inspection. The famer who owns these animals

have not bought in animals.

The full clinical findings as a result of examination by the ETC-5 team are shown in Table.1,

and the corresponding photographs are attached in ‘Annex 1’.

Clinical Results: Esenkent 04/05/2010

Vesicular lesion location/age

Animal ID

Species/sex

DOB/ Age

Clinical signs

Rectal temp (C)

LF RF RH LH Buccal cavity

Samples taken

Photo number (Annex 1)

Comments

Elevitkormaz (Barn 1)

Cow 1 Bovine Calf Salivation 37.8 None None None None N: 2-3 days T/DP: 5 days

Epi/P/S/B

Barn 1: ‘C1 – 1’ to ‘C1 – 10’

Primary vaccination (approx 3rd March) ?

Cow 2 Bovine Calf None 39.4 None None None None T: 10 days DP:7 days

P/S/B Barn 1: ‘C2-1’ to ‘C2-4’

Primary vaccination (approx 3rd March) ?

6443 Bovine Calf None 38.5 None None None None None P/S/B None Primary vaccination only (approx 3rd March) ?

Kadir Demirkaya (Barn 2)

TR 36153 9070

Bovine Female

27/06/09

Recumbent 39.8 None Pain ID, secondary infection

CB secondary infection. Surface skin temp. increased

None DP: 14 days T: 14 days

S Barn 2: ‘9070-1’ to ‘9070-9’

Vacc . 15/10/09. Also vaccinated approx 3rd March ?

TR 36168 6176

Bovine Female

21/07/09

None 39.4 None None None None DP: 14 days S/P Barn 2: ‘6176-1 to ‘6176-2’

Primary vaccination (approx 3rd March) ?

Vesicular lesion location/age

Animal ID

Species/sex

DOB/ Age

Clinical signs

Rectal temp

LF

RF RH LH Buccal cavity

Samples taken

Photo number (Annex 1)

Comments

Kazim Agdaf (Barn 3)

Cow 3 Bovine 4 months

Lame LF ID: 2-3 days

Non

e

None None None Epi/B/S/ Saliva

Barn 3: ‘C3-1’ to ‘C3-4’

Primary vaccination (approx 3rd March) ?

Atakan Demirkaya (Barn 4)

TR 36139 3931

Bovine Female

25/06/ 08

None None Non

e

None None DP: 10 days Tongue tip: 10 days

None (risk of abortion)

Barn 4: ‘3931-1’ to ‘3931-3’

Vacc. 01/11/09. Also vacc. approx 3rd March ? Heavily in calf

TR 36139 5224

Bovine Adult steer

02/05/ 08

None None Non

e

None Caudal CB separation

DP: 7-10 days Tongue:

S/Saliva Barn 4: ‘5224-1’ to ‘5224-2’

Primary vaccination (approx 3rd March) ? Late castrated.

Atalay Kuru (Barn 5)

TR 36148 8813

Bovine Female

12/12/ 08

Salivation/Lame

Medial claw CB under

As LF

Secondary bacterial infection coronary

Secondary bacterial infection coronary

DP: 6 days T: 10 days

Epi/S Barn 5: ‘8813-1’ to ‘8813-3’

Nasal lesion. Primary vaccination (approx. 3rd

-mined

band and separation heel bulb

band and separation heel bulb

March) ?

TR 36140 5058

Bovine Female

21/07/ 08

None None Non

e

Dorsal ID space: 1 day

None DP: 5 days T: 2-3 days

Epi/S/ Saliva/P

None Vacc. 20/04/09. Primary vaccination only .

Key:

Lesions: CB: Coronary band T: Tongue Samples: Epi: Epithelium P: Probang

ID: Interdigital space B: Blood

DP: dental pad S: Serum

Please see ‘Annex 1' for photographs of clinical findings

Table 2. A table showing the test results for samples collected from each animal examined in Table 1.

Serotype Antigen elisa results carried out on Epi

Animal ID Oldest lesion age

LFD Antigen ELISA O

Antigen ELISA A

Antigen ELISA Asia 1

NSP Multiplex PCR Probang

Multiplex PCR Saliva

Ecevitakormaz (Barn 1)

Cow 1 5 Pos 0.21 Neg 0.072 Neg 0.076 85/86Pos Positive - Type O N/A

Cow 2 10 N/A N/A N/A N/A 91/92 Pos Negative N/A

6443 No Lesion N/A N/A N/A N/A

64/61 Pos Negative N/A

Kadir Demirkaya (Barn 2)

TR 36 153 9070 14 N/A N/A N/A N/A 88/88 Pos N/A N/A

TR 36 168 6176 14 N/A N/A N/A N/A 86/85 Pos Negative N/A

Kazim Agadf (Barn 3)

Cow 3 3 Pos 0.134 Neg 0.076 Neg 0.056 92/92 Pos N/A Negative

Takan Demirkaya (Barn 4)

TR 36 139 3931 10 N/A N/A N/A N/A N/A N/A N/A

TR 36 139 5224 10 N/A N/A N/A N/A 91/89 Pos N/A Negative

Atalay Kuru (Barn 5)

TR 36 148 8813 10 Pos REPEAT + VI REPEAT + VI REPEAT + VI 90/91 Pos N/A Negatie

TR 36 140 5058 5 Pos > 3.00 Neg Neg 48/46 Neg Positive - Type O Weak Positive

0.1 OD value threshold for Antigen Elisa N/A indicates samples not taken

Fig.2. Esenkent predicted timeline of disease

The timeline shows the approximate dates of infection, and tracks the probable course of infection through the barns. It can be seen that from

the oldest lesion dates, that infection appears to have entered the village initially into Barn 2, then spread to Barns 1, 4, 5 (at approximately

the same time) and finally to Barn 3. This only represents our investigated barns as we do not have ınformation for the whole vıllage.

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Most likely incubation period

Incubation period

Pre-lesion virus shedding period

Clinical lesions

Sampling and testing for the presence of / exposure to FMDV

Samples taken depended on availability of sample and whether we were permitted to take

samples by the farmer.

Epithelium – Where loose epithelium was available, this was removed with forceps and

placed into virus buffer which was pH adjusted and was part of the LFD collection kit.

Blood – Blood collected into plain tubes was taken from the majority of animals.

Probang – Probang sampling was restricted to cattle where this was permitted by the

farmer, and also where clinical lesions were seen to be older.

Testing of samples

LFD and Antigen Elisa testing on the epithelium collected. This tested the presence of

viral capsid antigen in the sample (LFD and antigen elisa), and also serotyped these

viral antigens. The serotypes tested for were O,A, Asia-1.

NSP testing of serum collected. This tested for the presence of antibodies to viral

non-structural proteins (NSP) in the sample. Detection of these suggests viral

replication had occurred in this animal.

Multiplex PCR on Saliva and probing samples. This tested for the pesence of viral

RNA in the sample, and also indicated the serotype of the virus from which this RNA

derived from. The serotypes tested for were O,A, and Asia-1.

Cardiac Troponin T (cTnT) on serum samples. This tested for the presence of

circulating cTnT, which would indicate he presence of myocardial damage in the

animal. This was part of a pilot study.

Results of diagnostic tests

All results obtained are summarised in Table 2. Red indicates a positive result for the

corresponding test. Orange indicates an inconclusive result and will undergo further testing

at the SAP institute.

Discussion of diagnostic testing results:

Barn 1:

There were 3 animals on this barn. Two had clinical signs and one did not. They were all NSP

(non-structural protein antibody) positive. The cow with the freshest lesions had a sample of

epithelium taken, which was positive for LFD and for antigen elisa, Type O being the

suggested serotype from this test. Probangs were taken from all 3 animals, with only one,

the one with the freshest lesions, being PCR positive, again typed as serotype O from

multiplexing. The fact that all 3 cows were NSP positive suggests that all had significant virus

replication at least 4-8 days previously, if not longer (NSP correlation with onset of clinical

signs, personal observations, IAH Pirbright). The animal with no history of FMDV infection

(6443) may have been NSP positive for 2 reasons: One is that the test may have given a false

positive, however more likely is that this animal had been subclinically infected, without

showing clinical signs. This scenario is even more likely given the fact that this animal has

been vaccinated: subclinical infection of animals may occur in the face of vaccination against

FMDV.

Barn 2:

The two cattle infected here showed 14 day old lesions. No epithelium was available for

examination, and the probang collected from one of these animals was negative via PCR.

Serum samples were both positive for NSP. These results are expected form animals with 14

day old lesions, which have had time to mount an immune response to the NSP of the virus.

Viral clearance appears to have occurred in the pharynx of these animals.

Barn 3:

Cow 3

The animal examined in this barn had lesions aged at 3 days old. The epithelium sampled

was positive for viral antigen on the antigen ELISA, and suggested to be type O. Saliva

sample from this animal was negative on PCR. Interestingly, this animal was strongly

positive for NSP. Assuming the lesions ageing is correct, 3 days post-lesion onset is very fast

to develop a strong NSP response. This discrepancy may be due to the lesion age actually

being longer, in the range of 5 days +, or that this animal received a very large infectious

dose of virus, thus developing NSP antibodies much faster than normal.

Barn 4:

One of the animals in this barn could not be sampled due to it being pregnant. Both animals

examined had lesions of around 10 days of age. Serum from the one animals sampled was

NSP positive, with saliva sample being negative via PCR. These results are consistent with an

animal which has cleared virus infection and produced and thus produced an NSP response.

Barn 5:

Both animals examined in this barn were positive via LFD assay applied to collected

epithelium. Epithelium from the younger lesions (cow 5058) was positive via antigen ELISA,

which indicated Type O infection, whilst antigen ELISA result from the other cow (8813) was

inconclusive. The younger lesion (1 day) epithelium gave a much stronger response than

the older 10 day old lesion.

The serum from the 10 day old lesion cow (8813) was NSP positive, however that from cow

5058 (1 and 5 day old lesions) was negative. The high amount of antigen combined with the

relative young age of the lesion in cow 5058 suggests that the NSP negative result of the

serum is most likely due to the immune response not having enough time to mount a

response to the NS Proteins of the virus.

Discussion of Esenkent disease and vaccinal status

Information collected at the time of the visit from the farmers and from the local vet.

suggested that a vaccine was administered to all the animals in Esenkent on (approximately)

3rd March 2010. It was stated that no secondary/booster vaccination was given due to the

farmers’ perception of adverse reactions (milk drop).

From the ‘Turkvet’ database, it was noted that vaccine had been administered in October

2009/November 2009/April 2010 to three animals (see table), with no further vaccinations

recorded. However, the database has not been updated in some cases since the sale of the

animals; hence vaccination dates may be unrecorded. Reliable vaccination data is therefore

not available.

There has evidently been a failure of vaccination protocol in Esenkent. There has been

sporadic administration and recording, and the datasheet instructions have not been

followed (unwillingness to give a second booster for example). A solid immune response to

FMDV may not, therefore, have been illicited. The generally mild nature of clinical signs

noted in these animals may, however, be due to some degree of partial immunity.

A second possible cause of failure of protection in Esenkent is a lack of antigenic matching

for this serotype/sub-serotype. The vaccine administered was a trivalent killed vaccine (for

Serotypes O, A and Asia 1). The virus serotype was identified as Type-O via antigen ELISA

and PCR and thus most likely should be covered by administration of the trivalent vaccine.

There may be a possibility, however, that antigenic drift/mutation may reduce vaccine

matching to the outbreak strain, resulting in a degree of clinical disease (although this is less

likely than if the virus had been typed as serotype A). To investigate this data, generation of

R values by VNT, and ideally vaccine challenge experiments would need to be carried out.

However, the most recent evaluation of the current Type O FMDV virus circulating in the

area showed an r value of greater than 0.3 by VNT, which indicates sufficient vaccine strain

homology.

Ranking of risk factors for spreading of the disease

According to the result of the epidemiological investigation the major risk is the livestock

markets. This opinion is fully shared by the farmers.

Proper vaccination of cattle reduces the chance of infection, and if infection does occur,

clinical signs are normally not present, with a much reduced shedding and transmission rate

of disease to other animals. As vaccination protocol does not appear to be adhered to, this

is a major risk factor, making clinical infection and thus significant viral shedding and

transmission much more likely.

Once the disease is introduced in a farm, the herd management system, such as the practise

of common grazing, lack of inter farm biosecurity, provides a fast route of spread for the

virus to all animals in the village.

The surrounding villages may be involved in both the source and spread of infection via

direct or indirect contact (common grazing), or windborne infection. The involvement is

difficult to quantify in this case.

Important factors for stopping the spread of the disease

Improved vaccination administration, with rigorous and complete coverage of animals, with

an aim of eliciting a strong protective immunity in all animals.

Movement control should be reinforced, particularly ensuring that animals are moved

following veterinary examination and accompanied with the correct documentation.

Improve biosecurity and quarantine at both farm and village level.