# 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 -SENASA2

**EPIDEMIOLOGICA L SURVEILLA** **NCE IN FOOD A ND MOUTH DISEA** **SE FOOT A** **ND MOUTH DISEA SE SEROLOGICA** **L SA MPLING 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 situ ation 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 tech nicians of SENASA, of the National Agricultural Technology Institute (INTA), Animal Vi rology 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 (D i lacot). 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 v** **accination** ", the following sa m pling was ca rried out. **2. DESIGN**  **2.1 Objectives:**  2.1.1 To detect viral circulation of FMDV 2.1.2 Estimate 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 Sa m pling". The initial sampling is a sampling of two stages, whose population being studied i n cludes 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 ca rried out that consisted in the clinical inspection of susceptible animals, a study of animal move ments from the establishment, an epidemiological survey to the owner, and included a new serological sampling ("Supplementary Sampling"), with a specific d esign. **2 .** **2 Variables to measure:**  2.2.1 Presence of antibodies against non-structural proteins (PNE) of the FMDV (in suscepti ble species unde r 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 i m mune level. **2.3 Sampling Frame** **work :**  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 N o. 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 N o. 1) and the number of animals by herd that is necessary to include in the sample (formula N o. 2) was independently calculated.

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

##   

    



       

   2 1 1 1 1 e

N NC n r e r Where NC confidence (for the detection of at least one positive individual in the population ) e num ber of affected herds that can be detected as such in the population. This value com es from the mu ltiplication of the num ber 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 N r num ber of herds in the population to be studied

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

##   

    



       

   2 1 1 1 1 e

N NC n i e i Where NC confidence (for the detection of at least one positive individual by establishment) e num ber of individuals with the event, that can be detected by herd . This value comes from the mu ltiplication of the number of i ndividuals by herd (N i ) 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) N r 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 zon es , the number of sampled establishments in each zone was different according to the minimum5

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 fol lows:

-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 Reg ion 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 Bue nos 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 grazi ng, 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, a** **ccording 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 **TOTA L**  **265.708 55.168.494**  **2.414 19550**  (i) this was the only zone to wh ich 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 test** **s 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 sampli ng in non-vaccinated species). ELISA 3ABC and EITB system, according to procedures and specifications (kits PANAFTOSA/PAHO-WH O) 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 O 1 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 antibod ies 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. Fol**  **low -** **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 establish ment, 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 Sampli ng 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. **ZON E**  **N o. OF FA** **RMS**

**REQUESTED N**  **o. OF FA RMS**  **SA MP**  **LED %**  CENTRAL-MESOP 372 381 102.4% FATTENING 335 326 97.3% BORDER 459 456 99.3% NOA-CUYO 459 455 99.1% PATAG. NORT H A 330 329 99.7% PATAG. SOUTH AND NORT H B 459 459 100.0% **TOTA L**  **24 14**  **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 i n 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 Categor

y 2 (12 to 24 months of age ) by zone. **An imals Cat. 1**  **An imals Cat. 2**  **Zon e**  **An aly** **zed**

**Farms Positive** 

**Farms N**  **Positiv es**  **N Positiv** **es 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 **TOTA L**  **1941 37**  **1.9 11**  **578 24**  **0.2 7690**  **28 0.3**  The differences observed between the num ber 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 An**  **aly zed** 

**Farms Positive** 

**Farms An**  **aly zed** 

**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

- Wi nter 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: **Ty pe of farm**  **N %**  Breeding 54 66 Wi nter Fattening 6 7 Mixed 7 8.5 Dairy 4 5 Subsistence 3 4 Not categorized 8 9.5 **TOTA L**  **8 2**  **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 (S urveillance 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 anima ls 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 req uested 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. Posit** **i ve**  **Ovinos Pos**  **i tive**  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