**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 1 

### 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 ofthe Advisory Commission of Epidemiology, made up by a group of epidemiologists of official bodies dedicated to research such as the National Institute ofAgricultural 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 ofthe 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 ofsamples, 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 ofthe 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.**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 2 **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

- bothin 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.**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 3 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 ofAc against Non

-Structural Proteins), while ELISA flwas used in sheep/goats (detection ofAc against Structural Protein) for virus types A2001, O1 Campos and C3 Indaial and VIAA (non

structural 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**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 4 **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 designwere 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 ofa group of contact animals ofthe first lot.**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 5  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 ofviral 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 ofAc 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 ofELISA 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.**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 6 **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**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 7 As shown in figures 4 and 5, there are differences in the values obtained in both categories. This is due to the number ofdoses ofvaccine 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 ofthe 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 ofprotected sera at farm level in each ofthe 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.** C**onclusions:** 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 ofthe 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.**DNSA –**  **Dirección de Epidemiología y Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 8 For the second sampling stage, an expected protection of non-protected of 50%, is assumed because it is the value thatwill result in the maximum sample size.Assuming +/

- 0.1 (level oferror in the estimate) and an N original of4000 (number oflots), a sample of 100 lots or herds was indicated. The sampled animals were calves of6 to 12 months ofage that are part ofa 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 ofherds 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.