REPUBLIC OF ARGENTINA

NATIONAL SERVICE FOR AGRIFOOD HEALTH AND 

QUALITY 

EPIDEMIOLOGICAL SURVEILLANCE IN 

FOOT AND MOUTH DISEASE

SEROLOGICAL SAMPLING 2004

DIRECTORATE FOR EPIDEMIOLOGY

 NATIONAL ANIMAL HEALTH DIRECTORATE -SENASA
2

EPIDEMIOLOGICAL SURVEILLANCE IN FOOD AND MOUTH DISEASE

FOOT AND MOUTH DISEASE SEROLOGICAL SAMPLING 2004

1. Introducción:

SENASA annually implements serological samplings countrywide, whose objectives are 

both viral activity detection and estimates of population immunization due to FMD 

vaccination.

These samplings, with the other surveillance actions, serve to assess the epidemiological 

situation of FMD in the country and to obtain accurate indicators of its evolution.

The designs of these studies, as well as the evaluation of their results, is carried out in the 

Directorate for Epidemiology of the National Animal Health Directorate of SENASA, with the 

collaboration and technical assistance of the Epidemiology Advisory Committee, formed by 

technicians of SENASA, of the National Agricultural Technology Institute (INTA), Animal 

Virology Center (CEVAN) of Conicet and Veterinary Schools.

Sample collection is in charge of the local veterinarians of SENASA, while the processing is 

carried out at the Animal Laboratory of the SENASA Directorate of Laboratories and 

Technical Control (Dilacot).

Based on the criteria for the demonstration of absence of viral circulation to support the 

request to return to status as "FMD free zone with vaccination", the following sampling 

was carried out.

2. DESIGN

2.1 Objectives:

2.1.1To detect viral circulation of FMDV

2.1.2Estimate the immunity level against foot-and-mouth disease of the bovine population 

under the systematic vaccination program

The study for the detection of viral circulation was carried out through a work protocol that 

consisted in a first sampling, "Initial serological sampling" and, in the farms that resulted 

positive, a supplementary investigation which included a "Supplementary Serological

Sampling".

The initial sampling is a sampling of two stages, whose population being studied includes 

all agricultural establishments of the zone for which the return health status is requested.
3

The sampling was stratified into age categories and zones. In those establishments where

at least one reactor animal (positive) was detected, an additional research was carried out 

that consisted in the clinical inspection of susceptible animals, a study of animal 

movements from the establishment, an epidemiological survey to the owner, and included a 

new serological sampling ("Supplementary Sampling"), with a specific design.

2.2 Variables to measure:

2.2.1 Presence of antibodies against non-structural proteins (PNE) of the FMDV 

(in susceptible species under systematic vaccination program) and against 

structural proteins of FMDV (in non-vaccinated species), as an indicator of 

viral activity.

2.2.2 Antibody titer against structural proteins of the FMDV (structural antibodies) 

in cattle under systematic vaccination program, as indicator of the immune 

level.

2.3 Sampling Framework:

Sampling was implemented in the zone for which the return of the health status is 

requested.

Due to the large size, productive and geographical diversity of this zone, for design 

purposes was divided according to the following parameters:

- Animal density (cattle by ha)

- Composition of the cattle population: expressed as steer/cow ratio.

- Animal movement (entries and exits).

- Geographical and political-administrative characteristics.

- Risk zones (border zones).

- Zones next to free areas without vaccination.

With those criteria, the following zones were defined: (see Map No. 1)

a) Central - Mesopotamia

b) Fattening

c) NOA - Cuyo

d) Patagonia North A

e) Border

2.4 Sampled animals:

2.4.1 Bovines: sampled animals were stratified in the following age categories with the 

purpose to give temporality to the results:

- Category 1: 6 to 12 months of age.

- Category 2: 12 to 24 months of age.
4

Thus, the animals of category 1 reflect the last year’s sanitary condition, while those of 

category 2 reflect the last two years.

No samples of animals under 6 months of age are collected to prevent interference of 

calostrum antibodies.

2.5. Calculation of the sample size:

To determine the sample size required to detect whether an event is present in a 

population, the number of herds from where samples will be collected (formula No. 1) and 

the number of animals by herd that is necessary to include in the sample (formula No. 2) 

was independently calculated.

- Fórmula No.1: Number of necessary herds (nr)

  









 

 













  

2

1

1 1

1 e

n NC Nr

e

r

Where

NC confidence (for the detection of at least one positive individual in the population)

e number of affected herds that can be detected as such in the population. This value 

comes from the multiplication of the number of herds in the population to be studied (Nr) 

by the expected prevalence of positive herds and by the confidence that is defined for the 

determination of the number of individuals by herd to be included in the sample

Nr number of herds in the population to be studied

- Fórmula No. 2: number of individuals by herd to include in the sample (ni):

  









 

 













  

2

1

1 1

1 e

n NC Ni

e

i

Where

NC confidence (for the detection of at least one positive individual by establishment)

e number of individuals with the event, that can be detected by herd. This value comes from 

the multiplication of the number of individuals by herd (Ni) by the detectable prevalence, 

which is the result of the product between the expected prevalence (p) and sensitivity (Se) 

of the diagnostic method (N * p * Se)

Nr average of number of individuals by herd 

The number of establishments to sample is function of the confidence requirement, 

established assumptions, and the number of samples to collect in each farm.

Considering that, the confidence requirement (95%), the assumption of minimum expected 

prevalence of infected establishments (1%) and the number of samples to be collected by

establishment (for practical and operational reasons: 10), were common for all zones, the 

number of sampled establishments in each zone was different according to the minimum 
5

expected prevalence of infected animals by establishment (*) (inter-farm prevalence), and 

the average size of the herd. (See table no. 1).

The number of farms to be sampled in each zone was corrected according to the different 

inter-farm prevalence levels.

(*)The inter-farm prevalence estimated by zone (according to the productive characteristics 

and the historical behavior of the disease in each zone) was as follows:

-Central-Mesopotamia: 15%

-Fattening: 20%

-NOA - Cuyo: 10%

- Patagonia North A: 15%

-Border: 10%

2.6 Productive and epidemiological characteristics of each zone:

2.6.1 Central- Mesopotamia: 

- Location: it includes the Central and Mesopotamia Region of the FMD Eradication 

Plan 2001, with the exception of Fattening and Border areas (see item 2.6.2 and 

2.6.5). It is made up by the provinces of Chaco, Santiago del Estero, Misiones, 

Corrientes, Entre Ríos, Santa Fe, Córdoba, La Pampa, San Luis and Buenos Aires, 

with the exception of parties or departments that form the Fattening and Border zones.

- Type of production: more than 80% of the country's livestock population is in this zone. 

It is a very extensive and heterogeneous zone, with areas with optimal characteristics 

for agriculture and cattle fattening, and other marginal used for extensive cattle 

farming.

- Bovine density: variable, with values of 1 bovine per hectare (ha.) to 1 bovine every 10 

hectares.

- Steer/cow ratio: of 0.2 to 0.5

- Movements: the described heterogeneity determines that there is a significant number 

of cattle movements between establishments, within the same zone and towards the

fattening zones.

2.6.2 Fattening:

- Location: This zone is located in the center of the country, formed by the 

parties/departments of the northwest of the Buenos Aires province, northeast of La 

Pampa and south of Cordoba, Santa Fe and Entre Ríos.

- Type of production: Most establishments have production systems for cattle fattening, 

with intensive grazing, high stocking density, that determine high contact rates.
6

- Bovine density: is characterized by a high density of animals (1.5 bov / has.).

- Steer/cow ratio: 1.8

- Movements: it is an animal receiving zone, most of them coming from the main 

breeding regions of the country (Central-Mesopotamia and Border zone). Animal exits 

are, in general, for slaughter.

In this area an inter-farm prevalence higher than in other (20%) was estimated because 

due to the productive characteristics explained above, the high rate of contact between 

cattle determines a behavior different of the disease in case of occurrence (higher

probabilities of contagion).

2.6.3 NOA-Cuyo:

- Location: It is formed by the provinces of Salta, Jujuy, Catamarca, Tucumán, La Rioja, 

San Juan and Mendoza, except the border departments with Bolivia and Paraguay. It 

includes the Argentine Northwest (NOA) and Cuyo regions, of the 2001 FMD 

Eradication Plan.

- Type of production: Livestock farming in this zone generally is, extensive, extractive, 

and in some areas, subsistence, due to the poor quality of the soil and adverse 

weather conditions, with little possibility of calf fattening.

- Bovine density: 1 bovine every 20 hectares

- Steer/cow ratio: 0.2

- Movements: are generally rare; animals enter for slaughter and some for breeding.

In this zone, in case of disease occurrence, it is estimated to find a lower prevalence of 

infected animals by farm (10%) than in the central regions. This is due to the low livestock 

density that characterizes their livestock farms, with a very low rate of contact, as 

mentioned above.

2.6.4 Patagonia North A:

- Location: it includes part of territory bounded on the north by the Río Colorado and on 

the south by the Río Negro. It is formed by the Río Negro province area, where FMD 

vaccination is practiced (located north of the Río Negro) and the Patagones party of 

Buenos Aires province.

It was originally established within the 2001 FMD Eradication Plan as a buffer zone 

(with vaccination) for protection of the FMD free zone without vaccination (Patagonia 

North B and South Patagonia).

- Type of production: extensive livestock farming prevails, although there are small 

areas with artificial irrigation with fattening possibilities (Río Negro valley).
7

- Bovine density: 1 bovine every 4 ha.

- Steer/cow rate: 0.5

- Movements: due to their protective zone function, the entry of animals is very 

restricted, allowing only pure pedigree breeding animals, serologically negative to 

FMD, or for direct slaughter.

The assumption of inter-farm prevalence in this zone (15%) is similar to that established for 

Central-Mesopotamia, given that the productive characteristics are similar. However, due to 

the small size of this region, for the calculation of the sample size (establishments to be 

sampled), the formula was adjusted to the size of the population under study (1500 

establishments), so the number of establishments to be sampled was lower than in the 

other region (330 vs. 372). See table no. 1.

2.6.5 Border

- Location: it includes the northern border of the country and is formed by the 

departments of Jujuy, Salta, Formosa, Chaco, Corrientes and Misiones provinces that 

border Bolivia and Paraguay. It was defined because of the occurrence of foot and 

mouth disease in those countries during 2003.

- Type of production: the characteristics of this zone are not homogeneous, clearly

differentiating from east to west. Predominates to the east, the extensive cattle 

breeding establishments, while to the west, and as the rainfall regime decreases, 

there are marginal subsistence areas.

- Bovine density: in the east is 1 bovine every 3 ha., while in the west it is 1 every 30 ha.

- Steer/cow ratio: 0.3 in the entire zone

- Movements: is a zone with rare movements

- Number of farms and animals to be sampled by area, according to design
8

Table No. 1: Number of establishments and animals to be sampled, by zone for detection 

of viral circulation.

ZONE Number 

of Farms

Number of 

Bovines

Farms to 

be 

sampled

Bovines to 

be sampled

Central-Mesopotamia 166.845 39.982.910 372 3720

Fattening 35.701 10.620.908 335 3350

NOA-Cuyo 19.468 1.174.242 459 4590

Patagonia North A 3.152 993.530 330 (i) 3300

Border 39.018 2.363.311 459 4590

TOTAL 265.708 55.168.494 2.414 19550

(i) this was the only zone to which the size of the population was limited (3152 farms) for the 

calculation of the sample size.

Within each zone, the number of farms to be sampled by party or department was 

distributed proportionally to the bovine stock of each.

3. Diagnostic tests used

The study of viral circulation is carried out through the detection of antibodies against nonstructural proteins (NSP) of FMDV in cattle (vaccinated species) and antibodies against 

structural proteins (SP) in sheep (supplementary serological sampling in non-vaccinated 

species).

ELISA 3ABC and EITB system, according to procedures and specifications (kits 

PANAFTOSA/PAHO-WHO) is used for detection of antibodies against NSP.

Sensitivity (Se) and specificity (Sp) of the diagnostic tests (*):

Se: ELISA 3ABC 100% EITB 100%

Sp: ELISA 3ABC 97,97% EITB 99,27%

(*)Source: NSP Tests – Validation check list

For detection of antibodies against SP, ELISA is used in polyclonal fluid phase (ELISA fl), 

for viruses O1 Campos and A 2001.

The samples are analyzed at the SENASA’s official laboratory (DILACOT, Martínez), 

validated in proficiency tests by PANAFTOSA.
9

4. Sample collection

Sample collection in cattle was carried out during the first vaccination campaign of 2004. 

Sampling was carried out when applying the vaccine to reduce the possible interference of 

vaccine antibodies in NSP testing.

Population immunity given by vaccination, was assessed prior to the time of revaccination, 

which allows an estimate of the protection basal level of the bovine herd

5. Follow-up of establishments with reactor animals:

In those establishments where at least one animal reactor to the confirmatory test was 

detected, supplementary investigations were carried out that which consisted of:

a Visit to the farm, with clinical inspection of the susceptible population;

b. Survey to the owner and study of the movements of susceptible animals in 

the past year, according to the information recorded in the database of each 

local office;

c. Supplementary sampling:

Given that animals of the initial sampling were not individually identified, it was not 

possible to recognize them to include them in this supplementary sampling.

For this reason, a new sampling of a representative number of bovines 6 to 12 

months of age was carried out in the investigated establishments. To calculate the 

sample size, a confidence level of 95% and an assumed minimum prevalence of 5%, 

was established, for which 60 cattle were sampled by establishment, being identified 

with ear tags in this opportunity. Given that bovines from the same lot than those of 

the initial sampling were sampled, the inclusion of the latter in the supplementary 

sampling is not discarded.

Additionally samples of ovine or caprine were collected (sentinels species not 

systematically vaccinated), if they were present in the establishment, with the same 

sampling design that for bovines (60 animals), identifying them with ear tags.

Given that no animals of the species, categories and number specified in the 

investigated settlements were found, the number of samples in border 

establishments was completed.

When a positive reactor animal in the supplementary sampling was detected in some

establishment, a paired serology was made, carrying out a second serum extraction

in all animals of the group individually identified (cattle and sheep), with a minimum 

interval of 21 days, to detect a possible significant increase in magnitude and 

prevalence of antibody reactivity. Also, LEF samples were collected from reactor 

animals. 
10

Map No. 1: FMD sampling: Zonification

Note: The Patagonia South zone is a FMD Free Zone without Vaccination recognized by 

OIE; and in Patagonia North B vaccination is not practised, and is free of foot and mouth 

disease with surveillance function.
11

6. Results

6.1 Sampling compliance

Details the degree of compliance in the collection and delivery of samples, expressed in 

number of establishments sampled by zone, as has been requested according to the 

design.

Table No.1: Number of requested and sampled cattle farms, by zonea.

ZONE No. OF FARMS 

REQUESTED

No. OF FARMS 

SAMPLED

%

CENTRAL-MESOP 372 381 102.4%

FATTENING 335 326 97.3%

BORDER 459 456 99.3%

NOA-CUYO 459 455 99.1%

PATAG. NORTH A 330 329 99.7%

PATAG. SOUTH AND 

NORTH B

459 459 100.0%

TOTAL 2414 2406 99.6%

The non-compliance of the total of samples requested in some zones was due to:

• Problems in the transport to the laboratory: sample spills, broken tubes, poor 

preservation that made samples unusable, etc.

• Samples unfit for analysis (e.g. hemolysis).

These samples were rejected in the Reception Area of the Laboratory which is why they

were not entered or recorded in the data base.

The degree of compliance is satisfactory and statistically valid.

6.2 Results of Viral activity of the initial sampling

The following table shows the results of the tests for detection of antibodies against NSP 

obtained in bovines of the Category 1 (animals 6 to 12 months of age) and Category 2 (12 

to 24 months of age) by zone. Positive animal is the animal that resulted reactor to the two 

tests (ELISA 3ABC and EITB), and positive farm is where at least one positive animal was 

detected.
12

Table No. 2: Results to NSP tests obtained in bovines of the Category 1 (6 to 12 months of age) 

and Category 2 (12 to 24 months of age) by zone.

Animals Cat. 1 Animals Cat. 2

Zone

Analyzed 

Farms 

Positive 

Farms N

Positives

N

Positives

n % n % n %

Central -

Mesopotámica 380 10 2.6 2272 2 0.08 1510 11 0.7

Fattening 324 5 1.5 1942 2 0.1 1283 3 0.2

Border 455 8 1.7 2717 15 0.5 1818 3 0.16

NOA- Cuyo 453 5 1.1 2674 2 0.07 1782 3 0.17

Patagonia North A 329 9 2.7 1973 3 0.1 1297 8 0.6

TOTAL 1941 37 1.9 11578 24 0.2 7690 28 0.3

The differences observed between the number of samples entered to the laboratory and the processed ones 

were mostly due to non-conformities detected after their entry into the laboratory.

Table No. 3: Results to the SP tests obtained in ovine of Patagonia Ssouth and North B.

Zona

Analyzed 

Farms Positive 

Farms

Analyzed 

Serum 

Positive 

Serum 

Patagonia North B 100 1 (1%) 1000 1 (0.1%)

Patagonia South 359 0 3590 0

Total 459 1 (0.2%) 4590 1 (0.02%)

6.3 Follow-up of farms with positive animals in the Initial Sampling:

All farms with positive animals in the initial sampling had supplementary investigations, as 

developed in item 5 of this document

38 establishments resulted with at least one positive animal in the initial sampling. The 

distribution of these establishments by zones is detailed in tables 2 (see Map 1: location of 

the positive farms). Not all had the category, species or number of animals for the 

supplementary sampling; in those cases it was investigated, also a border farm, to 

complete sampling.

82 farms were investigated in total (38 positive and 44 border farms).

6.3.1 Epidemiological Survey: a survey was carried out to the owner or person in charge 

of the animals. Below is a summary of relevant data on them:

a) Type of farm: the establishments investigated were categorized in the following way 

according to their predominant production system:

- Breeding: livestock farm for calf production, which are fattened in other farms.
13

- Winter Fattening: production for cattle fattening that receives their calves from a 

breeding farm.

- Mixed (breeding and winter fattening): of full-cycle, produces and fattens their own 

calves.

- Dairy: establishment for milk production.

- Subsistence: type of production family farm, whose production is for their own 

consumption.

Distribution:

Type of farm N %

Breeding 54 66

Winter Fattening 6 7

Mixed 7 8.5

Dairy 4 5

Subsistence 3 4

Not categorized 8 9.5

TOTAL 82 100

b) Size:

The size of the establishments investigated was highly variable, with ranges of 10 ha to 

40,000 ha.

c) Size of the susceptible population:

The size of the susceptible population, by species was the following:

- Cattle: 76/82 establishments had cattle; the herd average was of 857 animals, with a 

range of 5-8474.

- Sheep/goats: 44/82 farms had ovine or caprine animals with an average of 177 

animals in each, with a range of 10 to 3500.

- Swine: 12/82 farms had pigs, with 2 to 32 animals in each.

d) Background of FMD vaccination:

All investigated cattle farms had complied correctly the last two vaccination campaigns, 

except that located in Patagonia North B (Surveillance Zone of the Region Patagonia 

South, without vaccination).

e) Background of FMD in the establishment:

A sampled establishment had background of the disease that occurred in May 2001.

f) Livestock feed system:

77% of the establishments (63/82) fed their animals only with natural pastures; 6 (7%) used

cultivate pastures and 6 (7%) used both pasture types. Only two establishments 

supplemented animals with grain.
14

g) Frequency of observation of the animals:

In 62 establishments (76%) the animals are observed daily.

h) Knowledge of FMD signs and regulations in force in regard to the disease:

In 76 cases (92%) owners or persons in charge of the animals had an adequate knowledge 

both about the symptoms of disease, as well as the obligation of denouncement in case of

suspicion.

i) Occurrence of confoundable diseases with foot and mouth disease in the last year:

In no case, signs confoundable with foot and mouth disease (lameness, drooling, decay, 

etc.) were observed in animals in the last year.

6.3.2 Supplementary serological sampling:

Another of the additional investigation actions that were carried out in the farms with 

positive animals in the initial sampling was the supplementary serological sampling, as 

detailed in item 5.3.

- Results: 38 farms were investigated. Given that not all had the category, species or 

number of animals requested according to the design, some boundary farms (44) were also 

investigated. Hence 82 farms were investigated, where 2,222 bovines and 1456 

ovine/caprine animals were sampled.

Positive reactors were detected in 4 farms, as follows:

Table No.4: Detail of samples collected and results of the 4 farms positive to supplementary 

sampling.

Farm Zone Bov. Sampled Ovine 

Sampled Bov. Positive

Ovinos 

Positive

1 Patag. North A 60 60 1 0

2

CentralMesopotamia 52 60 1 0

3 Fattening 60 29 0 1

4 Border 60 52 0 2

A paired serology was carried out in each of these 4 farms, carrying out a second sample 

collection of all animals of the group (cattle and sheep) with a minimum interval of 21 days, 

for purposes of detecting a significant increase in the extent and prevalence of antibody 

reactivity.

In the second collection, in three of these four farms all samples were negative, while in 

one of them the only serum which had resulted positive maintained its reactivity.

In addition, animals that had resulted positive reactors in this supplementary sampling, 

esophageal-pharyngeal fluid (LEF) samples were collected, where the virus was not 

isolated.
15

7. Conclusions:

1. The spacing distribution of the establishments with at least one positive reactor animal 

in the initial sampling did not required special studies by clusters of these.

2. The proportion of establishments with at least one positive animal, among zones, is 

almost homogeneous.

3. The analysis of the surveys carried out in the investigated establishments and the 

study of movements of these does not reveal a disease history in the last year.

4. The supplementary serological sampling indicated that the positive results of the initial 

sampling were due to non-specific reactions (false positive).

5. It is concluded that the results of this study provide reliable evidence that the viral 

circulation is not present in the zone under study