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

### E**pidemiological surveillance for**  **foot -** **and -** **mouth disease . Serological** 

**sampling Patagonia North A -**  **2013** The Epidemiology and Risk Analysis Directorate, branch of the National Directorate for Animal Health of SENASA, designed this study for the purpose o f complementing the information needed to request for the recognition of Patagonia North A as free of FMD without vaccination in accordance with the articles 8.5.42 to 8.5.47 and 8.5.49 of the Terrestrial Code of the World Organization for Animal Health (OIE). Hence the objective of this study is to demonstrate absence of FMD virus infection in the Patagonia North A region (part of the provinces of Río Negro, Buenos Aires and Neuquén ). In regard to the collection and delivery of samples, these were in charge of SENASA’s local veterinarians, whereas the diagnosis was carried out in the Animal Laboratory of the Directorate for Laboratories and Technical Control (DILAB

-SENASA). **Objetives :**  The objective of this study is to prove absence of infection by the virus of FMD (FMDV) in the zone of Patagonia North A. Infection indicators were assessed by the presence of antibodies against non- structural proteins (NSP) in cattle and against structural proteins (SP) in pigs (virus type A24 and O1 Campos). **Sampling fra mework** **:** This framework corresponds to the Patagonia North A zone, where there are about 600,000 cattle in 3,000 establishments, 430,000 sheep in 1,500 establishments, 45,000 goats and 25000 pigs. Annual serological samplings have been carried out in this zone since 2001. Pprobabilistic designs over the total of cattle farms, tending to demonstrate absence of circulation of FMDV at a detectable level of 1% of farm prevalence and 10% inter

-farm prevalence were used in these studies. The results obtained in all previous samplings were negative. A few years ago Argentina began the process aimed at achieving the recognition of the zone as free without vaccination; thus the last systematic vaccination campaign was carried out at the end of 2012, with the decision to suspend its definite application on March 4, 2013. As part of this process, it was considered appropriate to modify the sampling strategy in the zone, from a probabilistic sampling to sampling based on risk to prove absence of FMDV infection. In later years, this new methodology will lay the foundations of a system aimed to the early detection of an eventual entry of FMDV. Hence, it has been oriented to farms categorized as "risk", according to certain factors (farms that have a river shore, those where animals entered in recent years, vicinity of cold storage plants, garbage dumps,**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 2 among others), and it has been oriented to young cattle category (6 to 12 months of age) to detect if infection of the virus of FMD is present in PNA. Simultaneously with foot and mouth disease sampling in cattle in this zone, other surveillance activities were also carried out in pigs and wild animals to demonstrate absence of foot

-and

-mouth disease virus infection in these species. For this purpose, pig breeding family establishments adjacent to municipal waste dumps were inspected; carrying out clinical inspection to the animals, to which serological sampling was also performed. For this purpose, serum samples were collected for detection of specific antibodies against foot and mouth disease for virus A 24 and O1 Campos, also these samples were subjected to other diagnostic tests, such as Classical swine fever, Aujeszky, African swine fever, and porcine reproductive and respiratory syndrome ( PRRS) . Likewise in each case an epidemiological survey should have been completed. Also worked in collaboration with the provincial and municipal levels, encouraging meetings with intendants and staff of these Government spheres, in order to disseminate information of the surveillance work that was carried out and in this way achieve collaboration for the control and elimination of pigs from waste dump s, thus avoiding that these animals are fed with trash, waste and/ or slaughter remaining. As part of the surveillance activities, a survey and geo-referencing of open waste dump s was carried out and closer establishments with pig stock were identified. When animals were detected close to the waste dumps, these establishments were inspected and sampled. They also worked jointly with Provincial Wild Life authorities and Veterinary schools, Biological and Natural Sciences of the zone, in order to expand opportunities for sampling in wild animals susceptible to the disease. Wild boar samples were obtained from this surveillance in wildlife. **I .**  **Bovine Sampling: Sample size:**  A random probabilistic sampling in two stages was carried out; through the selection of establishments categorized as "risk" according to certain factors and the selection of cattle of the category 6 to 12 months of age . The assumptions determined were:

-Minimum expected prevalence of positive herds: 1%.

-Minimum expected prevalence of positive animals: 20%.

-Type II error: 5%.

-Confidence level: 95%.

-Sensitivity and specificity of the diagnostic test: 0.9. With these as sumptions described earlier, the number of sampled establishments was 245, with an amount of 15 cattle in each of the establishments, totaling 3.675 sera.**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 3 The underlying assumptions are: 1. The region was found free of FMDV infection, according to the samplings carried out annually for the past 12 years. 2. The probability of introduction of FMDV to the region since the last sampling is associated with various factors, among them certain establishments whose location or associated practices increased their risk in regard to the rest of the establishments of the region (risk establishments). 3. If infection is absent in the population at risk, then it is possible to accept that it is absent in the total population. 4. Establishments at risk were ident ified which were defined on the basis of certain criteria:

 Those establishments limiting the Río Colorado (n = 118), since the geographic proximity can be a greater probability of illegal entry of animals or by

-products.  Establishments with entry of breeding animals from the zone north to the Río Colorado (zone with vaccination) in the last two years (n = 402), since the movement implies the risk of entry of FMDV during the silent epidemic period.  Establishments limiting slaughterhouses, waste dumps, or auctions and fairs (n = 59), because they may be associated with exposure to contaminated products entered illegally; other factors of local risk as farms with a number of movements above the average and other factors for foot and mouth disease determined by the local veterinar

ian, were also considered. These classification categories are not mutually exclusive, since there are some farms with more than one risk factor; forming a subpopulation of maximum risk with the farms that have more than one risk factor that were sampled entirely (n = 23).**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 4 Figure 1. Geographical location of waste dumps, cold storage plants and auctions and fair of PNA

1 . The level of prevalence detection at farm level used in previous surveys (1% of farms) is maintained. 1 . If an entry occurs, the inter

-farm expected prevalence will be greater than 10% assumed for a vaccinated population. In the epidemic of Argentina in 2000

-2001, in nonvaccinated populations but with recently vaccination history, the inter

-farm average prevalence was 10%. Therefore, it is considered conservative to consider an intra

-farm expected prevalence of 20%. Thus, the sampling fraction represents 100% of the farms of maximum risk (with more than one risk factor, n = 23), 45% of the farms at risk (n = 245), and 8% of the total farms in the region (n = 2.950). The geographical location of the farms considered of maximum risk (with more than one risk factor) can be observed in Figure 2.**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 5 Figure 2. Geographical location of farms considered of maximum risk in PNA.

Category of sampled animals:

. The sampled animals were cattle of six to twelve months of age (category 1). In the case of those establishments that did not have the 15 animals of that category, the amount of samples with cattle of up to 24 months of age was completed. **Diagnostic tests:** All samples were processed at the central laboratory of SENASA ( Directorate for Laboratories and Technical Control, Martínez, Buenos Aires province), FMD Reference Laboratory of the World Organization for Animal Health (OIE).**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 6 Diagnostic tests carried out seek to detect the presence of antibodies against non

- structural proteins of FMDV (Elisa 3ABC/EITB System) in cattle as an indicator of infection In those establishments where at least one animal reactor to the confirmatory tests in the initial sampling (EITB) a supplementary study should be carried out in line with the recommendations of OIE, in article 8.5.49 of the Terrestrial Code; this supplementar y study aims to expand investigations, and confirm or discard the first result as an indicator of viral infection/circulation. **Results:** Figure 3 shows the geo-referenced distribution of the sampled premises farms. Furthermore, it is observed that these sampled establishments are in line with the highest concentration of cattle entry movements from establishments/auctions and fairs from the zone with vaccination to PNA.**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 7 Figure 3. Geo

-referenced location of the sampled farms and zones of highest

concentration of entry movements to PNA.

Table 1 shows the number of samples collected by cattle category, by local office: Table 1. Number of cattle sampled in the zone

**Province Local Office**  **Number of establishments**  **sampled Number of cattle** 

**sampled and analyzed of**  **Category 1 Number of cattle** 

**sampled and analyzed of Category 2**  **Total**

Buenos Aires C. Patagones 16 165 75 240

J. Pradere 13 76 134 210 Stroeder 20 206 94 300 Villalonga 19 244 41 285 **Subtotal 68**  **691 344**  **1. 035**  Neuquén Neuquén 3 30 14 44

Río Negro Choele Choel 31 456 9 465

Gral. Conesa 27 384 15 399 Gral. Roca 15 207 14 221 Guardia Mitre 17 255 0 255 Río Colorado 57 799 36 835 San Antonio Oeste 4 60 0 60 Viedma 15 218 7 225 Villa Regina 10 128 7 135 **Subtotal 176**  **2.507 88**  **2.595 Total**  **247 3.228**  **446 3.674** **DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 8 The results of all the bovine sera samples were "Non

-Reactive" to ELISA 3 ABC

-EITB, so it was not necessary to carry out supplementary studies.
**II. Sampling of pigs**  The serological study in pigs was supplementary to the survey of waste dumps carried out in the main populated centers. In this regard the visit of an establishment of family pig farming for inspection, survey and collection of blood samples was considered. **Sample size :**  To carry out this sampling, the number of samples to be collected was estimated, based on a simple random design for detection of an event in each visited farm. The assumptions used for each farm were:

-Inter

-farm prevalence of 20%. Expecting a relatively high prevalence in these populations because it involves non vaccinated animals and highly exposed to potential sources of infection due to the proximity to a waste landfill.

-Confidence level: 95%

-Type II error: 0.05 **Number of samples and category of animals**  **sampled :**  With the assumptions mentioned above for the number of samples to collect in each establishment it was completed according to the population present in the establishment, as shown in the following table: Table 2: Number of samples to be collected according to the establishment population

**Pig poulation of the establishment**  **N o. of pigs to be**  **sampled** <50 The entire

population 50 21 100 24 250 27 500 27 >1000 28**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 9 **Diagnostic tests:** All of the samples were processed at the central laboratory of SENASA (Directorate for Laboratories and Technical Control, Martínez, Buenos Aires province), The test used was ELISA fl. for detection of antibodies specific against foot

-and

-mouth disease virus (FMDV) for virus serotypes A 24 and O1 Campos. **Results:** The following table details the number of pig samples collected and analyzed, by local office: Table 3. Number of pigs sampled and analyzed in the zone, by local office of SENASA. **Province Local Office**  **Number of establishments**  **sampled N** **umber of pigs**  **sampled and analyzed**  Bs. As. Patagones 4 38 Neuquén Neuquén 14 68
Río Negro Choele Choel 16 95

Gral. Conesa 2 24 Gral. Roca 27 133 Guardia Mitre 4 37 Río Colorado 3 17 Viedma 6 50 **Subtotal** 58 356 **Total 76**  **462** All porcine serum samples tested were negative to ELISA fl. tests for both viruses. The survey analysis carried out in pig raising establishments that were sampled, adj acent to waste dumps, and the following information was obtained:
 76 establishments were visited, of which 58 belonged to Río Negro, 14 to Neuquén and the remaining 4 to Buenos Aires.  100% of the establishments belonged to a type of family production farm or subsistence. 78,7% of them had wiring, of which 13 were in good condition and 22 in regular conditioins .  The minimum and maximum total number of pigs by establishment was 1 to 98 animals.  The distance of the pig raising establishments in the risk areas are described in figure 4.**DNSA**
**– Dirección de Epidemiología y**  **Análisis de Riesgo -**  **Programa de Fiebre Aftosa** 10 Graphic 4. Distance of the establishments sampled to the identified risk areas

**Sampling of wild animals:** As it was previously explained they worked jointly with Provincial Wild Life authorities and the Veterinary Schools, Biological and Natural Sciences of the zone, in order to expand opportunities for sample collect ion in wild animals susceptible to the disease. Wild boar samples were obtained from this surveillance in wildlife . In 2013 f rom this surveillance in wildlife wild boar samples were collected distributed as described in table 4.
Table 4. Detail of the number of establishments and samples collected from wild boars.
**Province Local** 

**Office Number of** 

**establishments sampled**  **Number of wild** 

**boars sampled**  Buenos Aires C. Patagones 1 3* Río Negro Choele

Choel 5 10 Guardia Mitre 3 7 Río Colorado 1 1 **Total 10**  **21** *Only from one wild boar, serum could be extracted to be analyzed. These serum samples were analyzed by ELISA fl test for FMD virus serotypes A24 and O1 Campos, and were all found to be negative.