Foot-and-mouth disease

Outbreaks of FMD type C in Amazonas, Brazil

On 9 September 2004 the Ministry of Agriculture, Livestock and Supply of Brazil - MAPA - reported an outbreak of foot-and-mouth disease (FMD) in the Municipality of Careiro da Várzea, Amazonas (latitude 3º 13’ 77.6" south, longitude 59º 46’ 76.4" west).

The suspicion had been communicated to MAPA on 25 August by the owners of the operation, where a total of 4 bovines (1 to 2 years old) were reported to be affected. The animal population on the remotely located premises comprised 34 bovines, 15 sheep and 1 swine. The outbreak was located on an island in the Amazon River (Careiro Island), where animal movement is performed exclusively by river boat, and which is separated from the World Organisation for Animal Health (OIE)-recognized FMD-free zone with vaccination by more than 500 km. The results from the official laboratory in Belém, Pará, indicated FMD serotype C, which had not been reported in South America for almost ten years.

The Amazon region is enclosed in the northern livestock circuit, where the animal health and veterinary delivery systems are not as effective as those of other parts of the country. The area as a whole depends on the import of animals and meat from other parts of the country, and all livestock production is consumed locally.

The Brazilian authorities quarantined the affected premises and curtailed animal movement. An intense epidemiological surveillance was carried out around the zone; every farm on Careiro Island was quarantined, and five control posts and two surveillance boats were used to ensure no escape of the virus would occur.

Three secondary outbreaks were registered in the vicinity of the index farm, with 17 clinically affected bovids. On these premises there were 1 180 susceptible animals (973 bovines, 1 swine and 207 buffaloes). A total of 600 premises were visited and over 17 000 susceptible animals inspected. Every farm in the enclosed area was subjected to a new census and all bovine and water buffaloes (Bubalus bubalis) were vaccinated under official supervision. The Brazilian army, navy and local and federal government personnel involved in the activities included 12 veterinarians, 23 technicians and 78 assistants.

In Careiro da Várzea, 1 201 premises were subjected to supervised compulsory vaccination of a total of 70 477 bovines and 4 448 buffaloes. The epidemiological survey in Careiro da Várzea and neighbouring municipalities, including animal movement history, covered more than 17 000 animals without detecting clinical signs compatible with vesicular diseases. On 30 October 2004 all movement restrictions for animals and products were lifted. A minimum of two vaccinations was imposed for those animals entering the municipality.

Characterization of the causing agent by PANAFTOSA

Samples of the isolates of FMD virus (FMDV) type C were analysed by the Pan American Foot-and-Mouth Disease Center-Pan American Health Organization/ World Health Organization (PANAFTOSA-PAHO/WHO):

1. Typing and subtyping. Samples were processed at LAPA Laboratory in Belém with three passages on BHK (FC 50 percent), confirming the diagnosis of FMDV type C (registered as C3/Careiro da Várzea/AM/Bra/2004) (see Figure).
2. Immunogenic characteristics. A study on the expected percentage of protection (EPP) was carried out, using the sera bank at PANAFTOSA, collected from vaccinated and revaccinated animals with prototype vaccines of the region. The results showed EPP values of 52.15 percent at 30 days post-vaccination (DPV) and 95.91 percent at 30 days post-revaccination (DPR).
3. Typing genetic characteristics. BHK cell passage samples of the C virus sent by MAPA were submitted for molecular studies to be carried out at PANAFTOSA. Nucleotide sequence of the VP1 protein encoding gene were run and compared with type C reference strains. Results led to the conclusion that the isolate is historically endogenous to the continent. Nevertheless, it was not possible to establish a close relationship with any of the isolates of the PANAFTOSA-PAHO/ WHO data bank (maximum homology of 89 percent). Results of the comparison between the isolate C3/Careiro and C3/Indaial/Bra/71 (vaccine strain) showed a genetic difference of 13 percent in the region studied. This result precludes the hypothesis that the virus resulted from an escape from the vaccine industry.

Epidemiological study of the occurrence

Amazonas is included in an area considered by national authorities as of "unknown risk" for FMD. The state is presently in the process of improving its animal health services.

It covers an area of 1.5 million km2 (18.4 percent of Brazil’s surface) and has a bovine population of 1 152 108 head (0.6 percent of the country herd) and 11 950 farms (0.46 percent of the country). Production systems are family owned and operated, but some extensive farming also exists. Farming areas and animal movement are limited by the natural river tide (equatorial rain forest). Livestock produced is consumed within the zone, and additional livestock is supplied by imports from other states.

According to national authorities, disease detection capacity and vaccination levels are low in the region. The conditions for livestock production, the ecology, animal density and animal movement in the area, in addition to the difficulties faced by the veterinary services, are compatible with viral circulation and maintenance of FMD endemism in the bovine population. Genetic characteristics of the isolate of an indigenous virus support the fact that clinical activity was detected when surveillance capabilities of local veterinary services were improved.

Epidemiological analysis carried out by national authorities never excluded the possibility of diagnosing FMDV type C in the area, for which reason trivalent vaccines (O1, A24 and C3) are still used and relevant in the country.

Brazilian authorities have developed a phased national FMD eradication programme based on a regionalized strategy in accordance with the so-called "livestock circuits". As a result of this approach, Brazil has achieved recognition by OIE of the southern livestock circuit (Rio Grande do Sul, Santa Catarina and Paraná States), and eastern and western circuits (São Paulo, Mato Grosso, Mato Grosso do Sul, Goiás, Minas Gerais, Rio de Janeiro, Espírito Santo, Distrito Federal and Bahia States). During the processes of recognition a variety of aspects were taken into account such as epidemiological evaluations, analysis of vulnerability and receptivity, absence of clinical disease as well as viral activity, and vaccination coverage.

The free zone is physically separated from the endemic by natural barriers such as the Amazon jungle and wide rivers complemented with buffer zones. Additionally, animal movement into the zone is controlled by barriers staffed by federal personnel. The free zone is surveyed by 1 135 veterinary units, composed of 2 126 official veterinarians and 8 146 auxiliaries.

Brazilian animal health authorities have started to implement a FMD programme in the northern and northeastern circuits, which includes Amazonas.

It is accepted that the risk of type C virus spreading to FMD-free zones in Brazil and elsewhere in South America is very low mainly because of the absence of ecological and productive links with those areas and the enforcement of animal movement control activities. In the case of Brazil, with the exception of the State of Santa Catarina, where vaccination is not performed, the FMD-free zone with vaccination has a coverage that exceeds 90 percent with a trivalent vaccine (A, O and C).

Dr Eduardo Correa Melo, Director, PANAFTOSA-PAHO/WHO

Foot-and-mouth disease in Colombia

FMD was detected in cattle on a farm in Norte de Santander Department, Tibú Municipality, in August 2004. The outbreak was located approximately 16 km from the border of the Bolivarian Republic of Venezuela, in the OIE-recognized FMD protection zone bordering the FMD-free zone where vaccination is practised. Measures were undertaken to control the disease, such as destruction of the animals affected, quarantine, movement control, surveillance and ring vaccination. FMD virus type A, subtype 32, was isolated and characterized by complement fixation test. A second outbreak, affecting two pigs that resulted positive for FMD virus type A, was subsequently reported 0.5 km from the first outbreak. The last outbreak of FMD reported to OIE had been in September 2002. The 23 months without FMD reports from

Colombia likely represents the longest time the country did not have clinical disease since FMD was introduced over 50 years ago.

Above is a tree showing the relationship between A32 and A24 as well as other viruses from northern South America. The A32 subtype is represented by A Venezuela 70 (Bolívar, Bolivarian Republic of Venezuela), the predominant virus strain in the Bolivarian Republic of Venezuela from 1969-1980.

Dr Nick Knowles (Institute for Animal Health, Pirbright) has suggested that A32/Venezuela/70 is an antigenic variant of A24/Cruzeiro that may have arisen following the use of a live attenuated A24 vaccine in the region. He also noted that A32 has a one-way - virus neutralization (VN) and complement fixation (CF) - relationship with A22 Iraq. This is because the sequences of the G-H loop of VP1 are identical to each other and distinct from A24 - a case of antigenic mimicry.

Performance of serological tests for SAT-type FMD: a collaborative study in Zimbabwe

Serosurveillance can be used to investigate whether FMD virus circulation has continued after a disease outbreak has occurred, but knowledge about the performance of serological tests is crucial in the design of serosurveys. FAO recently undertook a collaborative study with the Zimbabwean Department of Veterinary Services to investigate the performance of serological tests, particularly in relation to SAT-type FMD virus infections.

Outbreaks of FMD in Zimbabwe provided an opportunity to carry out a field study involving the collection of specimens to evaluate different laboratory tests that had not yet been fully assessed with SAT serotypes. The primary objective was to assemble sera from cattle that had recovered from FMD in order to evaluate the performance of different serological tests in the serodiagnosis of SAT-type FMD virus infection. An additional objective was to compare the performance of test methods used to detect infection in vaccinated animals where cattle had been vaccinated and subsequently infected by SAT-type viruses.

Specimens were collected from FMD-convalescent cattle between 27 April and 7 May 2004 by staff at the Department of Veterinary Services, Zimbabwe, with assistance from the European Commission for the Control of Foot-and-Mouth Disease (EUFMD) Secretariat. These specimens were then sent to the FAO/OIE World Reference Laboratory for FMD (WRL FMD) in Pirbright, United Kingdom, for testing. This report outlines the study’s procedures and summarizes the preliminary laboratory data.

Collecting and processing samples

The initial plan was to "sample" at least 300 cattle at multiple outbreak locations in Zimbabwe. The criteria for herd selection were that clinical FMD had occurred between one and six months prior to collection and that convalescent or in-contact animals could be identified for sampling. In addition to collecting serum from each animal, it was agreed that other clinical specimens would be collected, including both oesophagopharyngeal (OP) fluid taken by probang cup and a swab from the nasopharynx^[2] and saliva^[3]. All necessary sampling materials were shipped to

Zimbabwe from the WRL FMD, along with packaging to enable the specimens to be returned safely and in compliance with International Air Transport Association (IATA) regulations.

The following protocol was applied at each sampling location:

1. Epidemiological information was gathered relating to the most recent FMD oubreak.
2. The identity of each sampled animal was recorded; dentition was examined to estimate age, and the hooves were examined for evidence of "linear breaks".
3. Specimens were collected in the following order: 50 ml of blood was taken from the jugular vein; two different types of swab were used to collect saliva; a guarded brush swab was passed via the nares to collect nasopharyngeal mucus/cells; and fluid from the oropharynx and oesophagus was collected with a probang cup.
4. A field "laboratory" was established at each sampling location to process specimens on site; saliva was expressed from swabs; and specimens collected from the pharynx (OP fluid and nasopharyngeal swabbings) were divided into aliquots for RT-PCR (to which a lysis buffer was added) and for virus isolation.
5. All specimens were labelled and were immediately stored on ice, with the exception of blood, which was allowed to clot at ambient temperature.

When they were brought back from the field, specimens were further processed at the Central Veterinary Laboratory, Harare. Serum (20-25 ml) was harvested from each clotted blood specimen, heat-treated for 30 minutes at 56 ºC and then stored at -80 ºC. All of the other specimens were placed in 2-ml cryotubes, sealed with parafilm and stored at -80 ºC.

Between 27 April and 7 May 2004, specimens were collected from 344 cattle at 5 outbreak locations in 2 different regions of the country. Linear breaks in the hooves of cattle at these locations were taken as an indicator of convalescence (from clinical FMD). The distance of these lesions from the coronary band, which allowed the time that had elapsed since infection occurred to be roughly estimated, was consistent with the epidemiological information gathered at each location. Virus isolation and typing had not been attempted on specimens collected from clinically affected cattle during the outbreaks at these locations. Therefore, at the time of sampling, it is was not known which virus strain or serotype was responsible for each outbreak. At some of the outbreak locations, cattle had been vaccinated with a trivalent vaccine (comprising SAT-1, SAT-2 and SAT-3 components) before and/or during the outbreak. Repeated vaccination may induce either serum antibody to non-structural proteins (NSPs) of FMD virus and/or FMD-specific IgA in saliva, which would be a complicating factor in the interpretation of test results. Therefore serum and saliva were collected from 60 cattle in a herd in which there had been no evidence of clinical FMD, but in which all cattle had been vaccinated on four separate occasions. All specimens were labelled, catalogued and stored as they were collected. When sampling was completed at all locations, a single shipment was sent to WRL.

Shipping processed specimens

For air shipment of specimens to WRL FMD the following steps were taken:

1. Arrangements were made with an airline accepting infectious substances (categorized by IATA as "dangerous goods"), as specimens for delivery to WRL must be sent by air freight direct to London Heathrow and not by courier.
2. Packaging was in compliance with IATA regulations, was sufficient to allow the inclusion of 50 kg of dry ice as a refrigerant and was well-insulated enough to keep specimens frozen for at least 48 hours.
3. A shipper’s declaration and other documentation (including labels) were completed in accordance with IATA shipping regulations and instructions provided by WRL (to ensure United Kingdom customs clearance).
4. A contact person at WRL was notified of the flight details and the airway bill number for the shipment.

Results

Preliminary laboratory data were recently presented to the Open Session of the EUFMD Research Group (Chania, Greece, 12-15 October). These data may be summarized as follows:

SAT-2 viruses were isolated from probang specimens collected from herds A and B (both located in the Mashonaland provinces of northern Zimbabwe) while SAT-1 viruses were isolated from herds C, D and E (located in Masvingo Province, southern Zimbabwe). Although FMD had not been observed in herd F, there was serological evidence to suggest that subclinical infection had occurred.

FMD virus was detected by one or more detection methods in 28 percent of the probang samples collected, the detection rate in different herds ranging from 14 percent (herd E) to 38 percent (herd C). However, the virus was rarely detected in nasopharyngeal swabs. One animal tested positive by virus isolation, and one by RT-PCR. Both of these animals were from herd D. Optimized RT-PCR (using custom-designed probes perfectly matching the genomic targets of viruses isolated at each location) was found to be more sensitive than virus isolation for detecting virus in probang samples. A standard RT-PCR protocol employing probes that had not been optimized for detection of these particular SAT isolates was of similar sensitivity to virus isolation.

The solid phase competitive ELISA (SPCE) and NSP antibody ELISA readily detected animals that had been infected with either SAT-1 or SAT-2 FMD viruses. NSP antibody ELISAs can therefore be considered as serotype-independent serodiagnostic tests that can be relied upon for the serodiagnosis of SAT-type FMD infections. Sensitivity estimates for the detection of FMDV "carriers" by NSP antibody ELISAs ranged from 75 to 90 percent with different ELISAs, a result very similar to the sensitivity values obtained with sera from experimental infections.

Summary of the sampled animals in each herd

* The number of animals in which linear breaks were apparent in one or more hooves/the number of animals examined; distance of linear breaks in the hoof wall from the coronary band

Acknowledgements

The authors wish to thank: Keith Sumption, Secretary, EUFMD, and Stuart Hargreaves, Department of Veterinary Services, Zimbabwe, for supporting this initiative from the outset; the European Commission (DG-SANCO) for financial support; Welbourne Madzima, George Gwaze, Nyasha Munjeri, Frank Chitate, Ann Muzira-Mujeyi, Maud Takawira, Mr Mponda, John Makwangudze, Thokozile Mswela, Ernest Dzimwasha and others at the Department of Veterinary Services, Zimbabwe, for their various parts in the field study; Geoff Hutchings, Nigel Ferris, Scott Reid, Andrew Shaw, Nick Knowles, Jean-Francois Valarcher, Satya Parida, Catherine Holmes, Debi Gibson, Mandy Corteyn, Rosa Fernandez and Pip Hamblin at the WRL, IAH, Pirbright, United Kingdom, for undertaking the laboratory testing of the specimens. A special mention must also be made of Wolfgang Boehle, FAOSAFR, Harare, Zimbabwe, for his cooperation and assistance with all of the practical elements of this field study.

Dónal Sammin, EUFMD Secretariat, FAO, and David Paton, WRL, Institute for Animal Health, Pirbright, United Kingdom