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FMD epidemiological Surveillance:

Serological samplings 2012

A) General sampling

Sampling framework

As a result of the foot and mouth disease occurrence in Paraguay and the Declaration of 

Health Alert in our country in September 2011, the serological surveillance was 

consolidated of 2012, in a study aimed to the Northern provinces that have international 

borders. For this, together with the first vaccination campaign in 2012 a serological 

sampling was carried out in the following six provinces, five of which have border with 

Paraguay: Salta, Formosa, Corrientes, Misiones, Chaco, and Jujuy, which was included to 

form part of the same region as Salta (Map 1).

The Epidemiology Directorate, dependent of the National Directorate of Animal Health of 

SENASA, designs these studies which are prepared with the collaboration of the Advisory 

Commission of Epidemiology, made up by a group of epidemiologists of official bodies 

dedicated to research such as the National Institute of Agricultural Technology - INTA-, 

the National Scientific and Technical Research Council - CONICET-, and colleges of 

veterinary medicine, among the most prominent.

Sampling is designed according to the standards and guidelines of the World Organization 

for Animal Health - OIE - both in its Terrestrial Animal Health Code, FMD Chapter, 8.5, 

Articles 8.5.43, 8.5.43.44 and 8.5.43.49, as in the Terrestrial Manual, Chapter 2.1.5, B. 

Diagnostic Technique.

The sampling operational part, as it is the collection and sending of samples, is carried out 

by official veterinarians of SENASA of the regional Office that has jurisdiction over the 

area being studied.

Diagnosis is carried out in the Animal Laboratory of the Directorate for Laboratories and 

Technical Control (DILAB-SENASA), which is a Regional Reference Laboratory for FMD 

(Americas) designated by the OIE.

Objectives:

a) To detect viral circulation or infection by the FMD virus (VFA).

b) To estimate the population immunity level for FMD in the cattle population of the zone.

The provinces included in this sampling are seen in Figure 1. 
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Figure 1. Map with the provinces included in the 2012 FMD sampling

Design:

Sampling is carried out following the guidelines of the World Organization for Animal 

Health - OIE - both in the Code and in the Terrestrial Manual. It was carried out based on 

the registration of establishments of the Animal Health Management System (SIGSA) of 

the 6 provinces described above.

For this a probabilistic, stratified design, was used in two stages. While for the calculation 

of the sample size the following assumptions were considered:

 Prevalence of infected herds: 1%

 Prevalence of infected animals: 10%

 Confidence level: 95%

On the basis of the above assumptions, 345 farms were selected, in each of which 28 

bovine were sampled, and in case of having non vaccinated species as sheep/goats; 

sampling from 10 animals was requested.

Sampled bovine were divided into two categories:

 Category 1: 6 to 12 months of age. 

 Category 2: 12 to 24 months of age.

While sheep/goats, should be less than 24 months of age.
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The farm selection was at random within the total number of establishments registered in 

the Animal Health Management System - SIGSA. In Figure 2 you can see the georeferenced location of the selected and sampled establishments.

Figure 2. Geographic location of sampled bovine establishments.

Diagnostic Tests:

ELISA 3ABC-EITB was the diagnostic test used in bovines (for detection of Ac against 

Non-Structural Proteins), while ELISA fl was used in sheep/goats (detection of Ac against 

Structural Protein) for virus types A2001, O1 Campos and C3 Indaial and VIAA (nonstructural proteins).

Results:

8.336 bovine sera and 1,698 sheep/goats sera, belonging to 345 selected establishments, 

were sampled and analyzed.

The results for detection of viral activity are described in table 1.

Table 1: Results for the detection of viral activity in bovines

Bovine 

category

Analyzed sera Reactors * %

Category 1 4.890 3 0.06

Category 2 3.446 0 0

Total 8.336 3 0.03

* Sera reactors to ELISA 3ABC-EITB
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Table 2: Results for the detection of viral activity in sheep/goats.

Analyzed farms Reactors* % Sampled Animals Reactors** %

183 0 0 1698 0 0

* Farms with, at least one reactor animal. 

** Animals reactors to ELISA fl /VIAA tests.

The location of the 3 reactor bovine belonged to three different farms, whose location can 

be seen in Figure 3.

Figure 3. Geographical location of the farms reactive to 3ABC-EITB ELISA tests.

The number of reactors obtained in this sampling is within the non specific values of 

diagnostic tests, and by location there is no evidence of clustering. Also, supplementary 

studies as it was intended in the design were carried out in the reactor farms.

Supplementary Samplings

With the emergence of a farm with any animal reactor to confirmatory tests, a 

supplementary follow-up study was carried out, according to the guidelines established in 

the OIE Terrestrial Code. The research consisted of:

 New visit to the farm to carry out a survey to the owner or keeper of the animals.

 Study of the movement of susceptible animal in the location (entries ad exit).

 New sample collection in order to carry out a supplementary serological study, in 

addition to a:

 Clinical inspection and new serological testing of the same group of animals 

from the first sampling if present.

 Clinical inspection and serological testing of a group of contact animals of the 

first lot.
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 Clinical inspection and serological testing of a group of non-vaccinated 

susceptible animals (sheep/goats), in case that they are available at that farm or 

at a neighboring farm.

 Extraction of esophageal pharyngeal fluid (LEF) for PROBANG of the reactor, 

if present. This sample is repeated in 21 days.

In none of the additional tests there was evidence of viral circulation or of disease, both 

clinical inspections as surveys, supplementary complementary serology or virological 

surveillance (LEF).

Immunity Studies:

According to the design, 13 samples were analyzed for immunity estimation by farm:

 10 sera of animals of 6 to 12 months of age (category 1).

 3 sera of animals of 6 to 12 months of age (category 2).

Of the total of samples sent, were selected:

 272 farms

 2706 sera of category 1

 815 sera of category 2

Diagnostic tests: 

The liquid phase ELISA (fl.) test was used for detection of Ac against structural proteins 

(SP) of the FMD virus for serotypes O1 Campos and A24 Cruzeiro, to estimate the level of 

protection.

The results obtained (values of ELISA fl.) for each serum, were categorized as "protected" 

or "unprotected". Were considered protected that serum that reached a level of Ac equal to 

or greater than the value that it corresponds with a Protection Percentage Expectation 

(EPP) of 75% for each serotype.

Figures 4 and 5 show the proportions of sera with protective titers for each category and 

province.
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Figure 4: Proportion of “protected” sera of category 1, by serotype and by province

Figure 5: Proportion of “protected” sera of category 2, by serotype and by province

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CHACO CORRIENTES FORMOSA JUJUY MISIONES SALTA

Inmunidad 2012

Categoría 1

% O

% A
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As shown in figures 4 and 5, there are differences in the values obtained in both categories. 

This is due to the number of doses of vaccine received in the animals from each category: 

in category 1 (from 6 to 12 months) may only have been vaccinated once, in the 2nd 2011 

campaign; while the animals of category 2 (from 12 to 24 months) may have received more 

than one dose, resulting in a higher antibody titer.

As sampling was immediately carried out before animals have been vaccinated in the first 

2012 campaign, it is considered that the immunity values that animals present, are the 

results of the previous campaign (post vaccination intervals exceed 5 months). Immunity is 

estimated in the animal population of the younger categories, which are the ones that have 

received less doses of vaccination, thus can be considered the least protected population.

Analysis:

Figure 6 details the distribution of protected sera at farm level in each of the provinces. As 

you can see, in all provinces except the province of Jujuy, which had few samples, there are 

a large proportion of farms where most of the animals are protected, correlating with the 

high values of protection individually observed.

Figure 6: Distribution of protected by farm, in each of the provinces included in the study.

Conclusions:

The immunity values observed in this study are satisfactory and are consistent with the 

high values of vaccine coverage achieved in 25 routine immunization campaigns carried out 

since 2002. 

In the population of adult animals, the findings of the study would be more accentuated 

due to their of multivaccinated population condition

A) Serological sampling in herds of the Border Line

This study was aimed to determine vaccine immunity for FMD in calf herds that are 

coming out from the border line, as category more susceptible to the disease (Resolution 

SENASA No. 44/2011, which regulates a special procedure for the movement of 

susceptible animals from the mentioned Border Line), and also sSera were analyzed to 

detect virus activity.

For sample size estimations, it was assumed that the immune status of the herd would be 

determined, with the objective of identifying non-protected herds at a 95% confidence, 

where "protected" is defined as a herd where at least 70% of the animals is protected. 

Therefore, the objective of the design is to identify, with 95% confidence, non-protected 

herds. The sample size was set at 15 animals per herd.
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For the second sampling stage, an expected protection of non-protected of 50%, is 

assumed because it is the value that will result in the maximum sample size. Assuming +/-

0.1 (level of error in the estimate) and an N original of 4000 (number of lots), a sample of 

100 lots or herds was indicated.

The sampled animals were calves of 6 to 12 months of age that are part of a herd coming 

out of the border line with a different destination that slaughter. For the purposes of 

distributing the study along the border, it was indicated to collect samples from 3 to 5 

herds by local office with jurisdiction over the border.

The results obtained are presented in the following table.

Table 1: immunity results for virus type A, O and C.

Sampled Farms/ 

herds

No. of 

sampled 

animals

Sera 

protected 

to Virus A 

(i)

Sera 

protected 

to Virus O 

(i)

Sera 

protected 

to Virus C 

(i)

104 1560 1162 

(74%)

1385 

(89%)

1290 

(83%)

(i) sera with greater titers than the cutoff value. 

At herd level, 5 of 104 (4.8%) resulted with insufficient amount of protected sera to a virus. 

The distribution of herds can be seen in the following map (in green the protected herds, 

and in red the non-unprotected herds).

Also all these sera were analyzed by the ELISA 3 ABC-EITB technique for detection of 

viral circulation, with negative result.