Semina: Ciências Agrárias ISSN: X Universidade Estadual de Londrina Brasil - PDF

Semina: Ciências Agrárias ISSN: X Universidade Estadual de Londrina Brasil de Paula Lyra, Tânia Maria; Pereira Freitas, Tânia Rosária Epizootiology, laboratory and virulence

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Semina: Ciências Agrárias ISSN: X Universidade Estadual de Londrina Brasil de Paula Lyra, Tânia Maria; Pereira Freitas, Tânia Rosária Epizootiology, laboratory and virulence analyses during the emergency phase of the African swine fever eradication program in Brazil in 1978: a historic account Semina: Ciências Agrárias, vol. 36, núm. 4, julio-agosto, 2015, pp Universidade Estadual de Londrina Londrina, Brasil Available in: How to cite Complete issue More information about this article Journal's homepage in Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative ARTIGOS / ARTICLES DOI: / v36n4p2577 Epizootiology, laboratory and virulence analyses during the emergency phase of the African swine fever eradication program in Brazil in 1978: a historic account Epizootiologia, análises laboratoriais e de virulência durante a fase emergencial do programa de erradicação da peste suína africana no Brasil em 1978: um relato histórico MEDICINA VETERINÁRIA Tânia Maria de Paula Lyra 1 ; Tânia Rosária Pereira Freitas 2* Abstract After the first African swine fever (ASF) outbreak occurred in Brazil in 1978, an official laboratory for ASF diagnosis (ASFDL) was established. The current work reviews the efforts of the laboratory team to define an ASF diagnosis during the emergency phase of the ASF Eradication Program. From June to December 1978, 3,803 samples of pig tissue, blood and serum were analyzed. ASFDL first isolated the ASF virus (ASFV) from pig tissue samples from the Teresópolis municipality in the Brazilian state of Rio de Janeiro. During the first two months, ASFV was isolated from 130 of 320 (40.62%) pig samples analyzed by haemadsorption (HAD), indicating that the outbreak had reached 96 of 214 municipalities surveyed throughout the country in that period. The distribution of positive ASFV samples indicated the potential route of virus dispersal. Because of the rapidity of the actions implemented against ASF, the number of ASFV positive samples decreased from 48.36% in June to 33.53% in July, and then to 0% in August In parallel, ASFV antibody detection increased from 17.89% in June to 52.04% in August In the state of Paraná, a comparison of ASFV isolates with descriptions of the disease, and a rapid decrease in mortality rates suggested the occurrence of a low or moderate virulence ASFV strain. Establishment of ASFDL was crucial for the program, which eradicated ASFV from the country within six years of the first outbreak. Advances in cellular and molecular biology corroborated pioneer studies regarding ASFV virulence, and have highlighted the importance of establishing and maintaining secure measures to prevent ASFV reintroduction to the country. Information on virulence heterogeneity in ASFV populations during outbreaks provided a paramount tool for the adoption of eradication best practices. Key words: African swine fever, epizootiology, haemadsorption, virulence, Brazil Resumo Após a ocorrência do primeiro surto de peste suína Africana (PSA) no Brasil, o laboratório oficial de diagnóstico de PSA (LDPSA) foi implantado. O trabalho atual revisa o esforço da equipe de laboratório para estabelecer o diagnóstico de PSA na fase emergencial do programa de erradicação. De Junho a Dezembro de 1978, 3803 amostras de tecido, sangue e soro de suínos foram analisadas. O primeiro isolamento do vírus da peste suína africana (VPSA) realizado pelo LDPSA foi feito em amostras de suínos oriundas da cidade de Teresópolis no Estado do Rio de Janeiro, Brasil. Em todo 1 PhD. Consultora em Defesa Agropecuária. Confederação Nacional de Agricultura e Pecuária do Brasil, CNA. Brasília, DF. Brasil. 2 PhD. Pesquisadora em Ciências Exatas e da Natureza. Laboratório Nacional Agropecuário, LANAGRO/MG, Ministério da Agricultura, Pecuária e Abastecimento, MAPA. Pedro Leopoldo, MG, Brasil. * Author for correspondence Recebido para publicação 10/04/14 Aprovado em 16/05/ Lyra, T. M. P.; Freitas, T. R. P. país, nos primeiros dois meses, de 320 amostras analisadas, 130 (40.62%) foram positivas pelo teste de hemadsorção o que sugere que a infecção com VPSA alcançou suínos em 96 dos 214 municípios analisados no período. A distribuição dos isolamentos do vírus no país mostrou uma possível rota de dispersão viral. Devido à rapidez das ações de eliminação dos focos, o número de amostras positivas caiu de 48,36% em Junho para 33,53% em Julho até nenhuma em Agosto, de Paralelamente, a detecção de anticorpos aumentou de 17,89% a 52,04% de Junho para Agosto de Os isolamentos do vírus pela técnica de hemadsorção (HAD) quando comparados com a descrição da doença e o forte declínio na taxa de mortalidade no Estado do Paraná sugeriram a ocorrência de cepas virais de VPSA de baixa a moderada virulência. A implantação do LDPSA foi crucial para o programa que assegurou a erradicação do VPSA em todo país após seis anos do primeiro surto. Os avanços na biologia celular e molecular corroboraram as suposições dos estudos pioneiros sobre a virulência viral e alertam para a importância de se estabelecer e manter medidas de segurança para prevenir a reintrodução do VPSA no país. A informação sobre heterogeneidade na virulência das populações virais nos surtos fornece uma ferramenta fundamental para a adoção de melhores medidas de erradicação. Palavras-chave: Peste suína africana, epizootiologia, hemadsorção, virulência, Brasil Introduction African swine fever (ASF), first described in Kenya in 1921 (MONTGOMERY, 1921), is a hemorrhagic viral disease that affects wild and domestic pigs of all breeds and ages. The high morbidity and mortality rates associated with ASF and the lack of preventive vaccines make the disease one of the major causes of economic losses to pig farmers, and a central issue for global food security (ARZT et al., 2010). ASF infection of domestic pigs can occur in peracute, acute, chronic and subclinical forms, depending on viral strain virulence and host factors (NEILAN et al., 2002). Initially endemic to sub-saharan African countries, the ASF virus (ASFV) has spread rapidly into countries never before infected (BASTOS et al., 2004). In 2007, ASF reached Georgia in the Caucasus region before spreading to neighboring countries (PENRITH; VOSLOO, 2009). The first official notice of ASF in Russia was in November 2007, and since then more than 900,000 pigs have died (MALOGOLOVKIN et al., 2012). Accumulated worldwide losses from ASF were an estimated US$1 billion in 2012 (World Organization for Animal Health OIE, 2012). The FAO and the OIE have warned of the risk of ASF reaching China, and have conducted outbreak simulations in several countries. The African Swine Fever Virus (ASFV) is a complex icosahedral, cytoplasmic, linear doublestranded DNA, lipoprotein-enveloped, arbovirus from the Asfarviridae family, Asfivirus genus (DIXON, 1986; IYER et al., 2006). The genome of several ASFV strains has been completely sequenced and consists of a linear double-stranded T-A rich DNA molecule ranging between 170 and 190 kbps, depending on the isolate, and containing hairpin loops as well as terminal inverted repeats (CHAPMAN et al., 2008). High virulence virus strains cause ASF in the peracute and acute forms, which develop into a severe acute hemorrhagic fever with death rates nearing 100% in 2-10 days, sometimes even before clinical signs are observed and an antibody response is mounted (MAC DANIEL, 1981; MEBUS et al., 1981). Sub-acute, chronic and unapparent forms of infection are associated with low virulence strains, which elicit antibody production (LYRA, 1980). Subclinical ASF infections are difficult to distinguish from classical swine fever (CSF) (MEBUS; SCHAFLER, 1982). Chronic ASF infection occurs in a variety of forms and can persist for several months without clinical signs. Recovered ASFV-carrier pigs and persistently-infected pigs pose an obstacle to disease control (HESS, 1981). European wild boars and feral pigs have similar clinical signs and mortality rates as those seen in domestic pigs (OURA et al., 1998). Argasidae ticks of the Ornithodoros genus, which infest the African wild pig warthogs (Phacochoerus africanus), play an important role in 2578 Epizootiology, laboratory and virulence analyses during the emergency phase of the African swine fever eradication... maintaining ASFV in nature (BASTO et al., 2006; BOINAS et al., 2004). In Brazil, the first reported ASF case occurred on the Floresta pig farm in the municipality of Paracambi, Rio de Janeiro, on May, 1978 (LYRA et al., 1986). The epidemiological, economic and surveillance aspects of ASF outbreak eradication have been described by Lyra (2006). Extending this previous study, we review here the efforts of the Brazilian ASF Diagnostic Laboratory (ASFDL) to establish the ASF diagnosis during the initial six-month emergency phase of the eradication program (1978), when the ASF virus was isolated in several outbreaks (MINISTÉRIO DA AGRICULTURA, 1984). We emphasize the aspects of ASFV isolation and dispersion throughout the country, which indicated that the Brazilian ASF outbreaks were caused by an ASFV strain of low to moderate virulence. Materials and Methods ASF occurrence data ASFDL registration data were acquired from the former National Animal Reference Laboratory (LANARA). Some of the data were provided by the libraries of the Rio de Janeiro and Minas Gerais Federal Universities in Brazil. Several official documents from Dr. T. Lyra s personal files were also consulted. Pig tissue and serum samples were collected from registered pig herds with at least one pig presenting suspicious clinical signs of ASF (LYRA et al., 1986; MACHADO JUNIOR, 1990). From June 1978 to April 1982, 41,829 samples were collected from pigs from the Northern, Northeastern, Central, Southern and Southeastern regions of Brazil. From June 12 to December 28, 1978, 3803 pig tissue, blood and serum samples were sent to ASFDL and were identified by number, arrival and test dates, municipality, state, farm ownership, and health inspector s name. We also investigated 559 epidemiological surveillance records (FORMS): 283 filled out between June and December 1978; 158 during 1979; 82 during 1980, and 36 during To understand ASFV dissemination in the state of Rio de Janeiro, municipalities where ASF outbreaks occurred were geographically distributed in mesoregions for proximity and cultural grouping. ASFV spread from the Southeastern to other regions was visualized on a map of Brazil on which all municipalities affected by ASF were plotted. ASF diagnosis data During the first month of its establishment, between June 12 and July 10, 1978, ASFDL implemented ASFV laboratory diagnostic practices following OIE Standards (Office International des Epizooties World Organization for Animal Health). The laboratory detected virus-positive samples using haemadsorption (HAD), a technique that relies on the property of infected leukocytes to induce erythrocyte adsorption. In parallel, the ASFDL team prepared suspensions of pig organs and tissues preferentially targeted by the virus such as tonsils, lymph nodes, spleen and liver. Each tissue suspension was inoculated on swine leukocyte cultures, and 24 to 72h later a swine erythrocyte suspension was also inoculated on the leukocyte cultures (HESS; OETRAY, 1960; MALMIQUIST, 1962). The fluorescent antibody test (FAT) or direct immune fluorescence (DIF) techniques were used to detect the ASFV antigen in tonsils, lymph nodes, spleen, and liver by impression smears of tissues, or on buffy coat cells on glass slides (BOOL et al., 1969). Immunoelectroosmophoresis (IEOP) was the standard serological test extensively applied to screen for antibodies against ASFV in pigs from different regions (ANDRADE, 1980; PAN et al., 1972). Implementation of CSF virus (CSFV) differential diagnosis began on July 10, Pig tissue suspensions were inoculated in the porcine kidney cell line PK15, and CSFV was detected 24, 48 and 72h post-inoculation by indirect immune fluorescence (IIF). The CSFV diagnosis was also performed by FAT. Dr. J. L. H. Facchini and his group in the Parasitology Department of 2579 Lyra, T. M. P.; Freitas, T. R. P. the Rural Federal University of Rio de Janeiro (UFRRJ) intensively screened for Argasidae in pig smallholdings with inferior housing facilities and poor sanitary conditions in Rio de Janeiro, where several ASF foci had occurred. Fortunately, they did not detect soft ticks of the genus Ornithodoros in pigs, which made possible complete elimination of the disease without the persistence of viral reservoirs in the ecosystem. ASFV isolation, disease description and mortality rates in the state of Paraná The FORMS filled out during the emergency phase of the ASF eradication program in the state of Paraná (PR) registered 59 pig herds from 43 municipalities during June and July of From these herds, 58 pig samples were tested by HAD. In June and July, 1978, respectively, 11 and 9 FORMS with one pig herd/form registered the isolation of ASFV. Number of passages needed for ASFV isolation by HAD, description of ASF pathogenic signs, and mortality rates were compared for the characterization of heterogeneity in the viral population. Results and field information from the PR municipalities where ASF was surveyed and ASFV was isolated by HAD were plotted on a map. Results and Discussion Distribution of ASF outbreaks in Brazil The ASF combat strategy that was employed can be divided in two phases: the emergency phase, from 1978 to 1979, and the specific program, from 1980 to 1984 (MINISTÉRIO DA AGRICULTURA, 1984). The ASF emergency phase started immediately after the first outbreak occurred in the municipality of Paracambi, Rio de Janeiro. Several ASF experts from Europe and North America were invited to collaborate in setting up a laboratory for diagnosis of ASF. Drs. William Hess, Keith Shermann and Joe Kresse (Plum Island Animal Disease Center PIADC) joined the efforts to establish ASFDL, and Drs. I. C. Pan (PIADC) and A. Ordás (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria INIA) contributed to the serology work (ANDRADE, 1980). In spite of the efforts that led to quick establishment of ASFDL, samples from distant regions sometimes deteriorated before arrival because of the poor transport and communications systems available in In those cases, the ASF surveillance team had to revisit sites and take new samples, inevitably delaying laboratory results. ASFV was investigated by HAD in different types of samples, including tonsils, lymph nodes, spleen, liver and/or blood. FAT was performed to detect ASFV antigen in tissues and to distinguish the ASFV cytopathic effect (CPE) from that of other swine viruses such as herpesvirus (Aujeszky disease virus). However, FAT lacks sensitivity to detect ASFV antigens associated with sub-clinical and chronic infections, because the antigen-antibody complexes formed in infected tissues block the interaction between virus antigen and antibody test indicator. Thus, HAD was considered the definitive test. The first Brazilian ASF outbreak occurred in the Floresta pig farm, a rustic smallholding in Paracambi, Rio de Janeiro, where pigs often fed on discarded food. Moreover, pigs from different breeds and ages were raised together and confined in a simple house, and the occurrence of other diseases such as salmonella was not uncommon. Most pig farms where ASF outbreaks occurred maintained similar sanitary conditions (LYRA, 1981). Before the ASF diagnosis was confirmed, the owner of the Floresta pig farm has sold pigs and pig products in several establishments in different municipalities in the state of Rio de Janeiro. His trade activities contributed to the dispersion of ASFV within the state (MACHADO JUNIOR, 1990). A commercial establishment in the slum community Nova Brasília in the city of Rio de Janeiro, where the second ASF focus was detected in early June, had acquired live 2580 Epizootiology, laboratory and virulence analyses during the emergency phase of the African swine fever eradication... pigs from the Floresta farm (MACHADO JUNIOR, 1990). In Nova Brasília, a sow with ASF clinical signs was found scavenging in the community garbage. It was sacrificed and samples were also sent to PIADC where the ASF disease was reproduced and the virus isolated (MACHADO JUNIOR, 1990). On June 12, 1978, the first five pig spleen samples arrived at ASFDL for ASF diagnosis. These samples were collected from a sick pig that had fed on a garbage dump in the municipality of Teresópolis. Machado Junior (1990) reported that infected carcasses in the garbage probably originated from the Floresta farm pigs. Two of these samples became the first ASFV isolations performed by the ASFDL team under the supervision of international advisers (Table 1). Table 1. ASFV diagnostics in the state of Rio de Janeiro between June 12 and July 10, ASF-HAD-positive samples are distributed by municipality and geographic mesoregions. Municipals of Rio de Janeiro Geographic State (RJ) Mesoregion Distance from Paracambi HAD Positive Teresópolis Metropolitan 102 km 02 Campos North N 319 km 08 Alcântara Metropolitan 91 km 01 Maricá Metropolitan 113 km 03 Itaguaí Metropolitan 33 km 01 Itaboraí Metropolitan 103 km 01 Duque de Caxias Metropolitan 57 km 02 Niterói Metropolitan 88 km 03 Bom Jesus Itabapoana North-East NE 316 km 03 Barra do Piraí South S 24 km 02 Carmo Center C 155 km 01 Total: Although Teresópolis is located 102 km from Paracambi, the two cities were directly connected by roads in 1978, and both are in the same Rio de Janeiro mesoregion. Most ASFV-positive pig samples in the state of Rio de Janeiro came from municipalities easily connected by roads in the Metropolitan Mesoregion, which had intensive truck traffic (Figure 1). Positive samples were also later detected in Barra do Piraí (South Mesoregion), Carmo (Central Mesoregion), Bom Jesus de Itaboapana (Northeastern Mesoregion), and Campos (Northern Mesoregion) (Table 1 and Figure 1). All of these samples came from farms that maintained a rustic rearing system where pigs frequently fed on meat swill. In face of the ASFV persistence in pork products, and the road trade connection of the initial outbreak sites with Paracambi, it is almost certain that the ASF outbreaks in the state of RJ had the same origin in the Floresta farm. The risk of virus dispersion led investigators to expand virus screening to the states of São Paulo (SP) and Minas Gerais (MG) in the Southeastern region; Paraná (PR), Santa Catarina (SC) and Rio Grande do Sul (RS) in the Southern region and other states in the Northern and Northeastern regions of the country. In June 1978, 74 (48.36%) of 153 pig samples tested were ASFVpositive (Table 2), including 28 and 7 positive outcomes at the second and third passages in pig leukocyte cultures, respectively. 2581 Lyra, T. M. P.; Freitas, T. R. P. Figure 1. ASFV spread through geographic mesoregions of the state of Rio de Janeiro. The first ASF outbreak occurred in the Metropolitan mesoregion and was disseminated to the others by road. Black lines: main roads connecting cities. Mesoregion symbols: M, Metropolitan; N, Northern; NE, North Eastern; C, Central; S, Southern; LC, Lower coastal. Table 2. ASFV diagnostics in Brazil during the emergency phase of the eradication program: ASF detection by HAD, FAT and serological analysis from June to December 1978 from the entire country HAD FAT IEOP Sample Sample Positive/ Negative/ Positive/ Negative/ Positive/ code received Tested Tested Tested Tested Tested June /153 79/153 71/143 72/143 17/95 July / /167 53/ /163 32/102 August / /171 1/ / /196 September / /155 0/88 88/88 04/179 October / /1
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