Specific diagnostic and epidemiological procedures adopted for the contagious
bovine pleuropneumonia eradication programme (1995-2002) in Portugal:
A working model for the disease control in African countries

José Regalla

Laboratório Nacional de Investigação Veterinária, Estrada de Benfica 701,
1549-011 Lisboa, Portugal

Introduction

Contagious bovine pleuropneumonia (CBPP) has re-emerged in a few European countries during the last 10 years. This severe contagious disease causes important economic losses and considerable socio-economic problems in many countries worldwide, espacially in Africa. The control of diseases with such impacts is an important objective of National Veterinary Administrations.

Evolution of the disease

CBPP was confirmed in Portugal in 1983 after being absent from the country for about thirty years (1954-1983). It was first recognised in January in a tuberculin positive dairy cow slaughtered at the abattoir of Monção, north of Portugal, near the Spanish border (Regalla, 1984). The disease became endemic in the coastal strip of the agricultural regions of Entre-Douro e Minho (EDM) and Beira Litoral (BL), where the intensive dairy farming is practised and the production of milk is most significant. Besides socio-economic features the most important factors responsible for the spread of the disease and the endemic situation in both infected regions of EDM and BL were the very high densities of bovine population and common milking parlours, as well as the very high numbers of farms very dependent of external replacement. Other important factors were the existence of fairs, traditional cattle-shows and communal pasture lands.

A national serological survey, carried out in 1983 provided an assessment of the incidence and prevalence of the disease, demonstrating a progressive increase from the South to the North with a high number of outbreaks occurring in the central Northern area of the country in the regions of EDM and BL and sporadic outbreaks in Trás-os-Montes (TM) (Regalla, 1984). For disease control and eradication between 1985 and 1989 the country, was divided into three different areas, Endemic, Risk and Disease-free Zones. From 1989 a new reinforced eradication plan was established, co-financially supported by the European Community, and the country was divided into Infected, Buffer and Disease-free Regions (Regalla et al., 1996). The following programmes have been developed and adapted according to the epidemiological progression of the disease. A wide programme for 5 years (1985-1989) and further, programmes of 3 and 2 years were established: (1990-1992); (1993-1995) and (1996-1997).

The main measures applied between 1985 and 1989 were the following:

i) Endemic Zone, serology on all holdings once a year and ban on bovine movement out of the area;

ii) Risk Zone, serology, once a year, on about 50% of random holdings and control of bovine movement inside the zone;Disease-free Zone, serology, once a year, on 15% of randomly chosen holdings and bovine movement controlled, only allowed from holdings of risk zone where no cases of CBPP or even positive serological results had ever occurred. Further developments lead to changes in serological monitoring during the period between 1989 and 1997. In the Infected Region, serological surveys were increased to twice a year on all holdings and in the Disease-free Regions, but only on 10% of random selected holdings. All sero-reacting animals were slaughtered, lungs and lymph nodes examined and lesions submitted to laboratory investigations. Two other systems were developed to complement the sero-surveillance: (a) abattoir inspection of routinely slaughtered beef and dairy cattle to detect lung lesions suspicious of CBPP. If infection was confirmed an effective trace-back system was installed to allow the identification of the infected herd; (b) the surveillance of suspicious clinical cases at herd level. All sero-reacting and suspicious clinical cattle were slaughtered, their lungs and lymph nodes examined and the lesions submitted to laboratory investigations. Stamping outs, involving thousands of animals, were made during 1997 in the areas of highest disease prevalence in both regions EDM and BL.

A decreasing trend in CBPP outbreaks was seen in EDM and BL since 1986: 466 in 1986 against 12 in 1998. In EDM the total number of outbreaks in 1998 correspond to a prevalence of 1.1 outbreaks in 10,000 herds. The last outbreak in Portugal was reported in February 1999 where, at the time 91,216 herds were recorded and occurred in the EDM region. Since then no outbreaks have been reported. In EDM, the most affected region, between 1989 and 1999, the decrease in the number of cattle with lesions was significant: 1,141 out of 2,485 had lesions in 1989 versus 19 out of 1,747 in 1998. In 1999, only 1 animal out of 191 slaughtered showed typical lesions.

Adopted tests

Until 1997, within the framework of the eradication programmes, the methodology used for CBPP diagnosis involved the following tests: (i) Complement fixation test; (ii) Bacteriological examination of suspicious material and, (iii) Histological examination of lesions. From 1997, besides the conventional tests, Polymerase Chain Reaction was also been applied (Bashiruddin et al., 1994).

The CFT was an essential tool of the eradication campaign with a high specificity of 99.5% but during the last years (Regalla, 1995), due to the low prevalence of the disease, its specificity became critical with false positive results corresponding to about 5,000 cattle slaughtered per year. As a consequence, a strong research effort to improve the test methodology was carried out, culminating with the successful development of a new test: the Immunoblotting test (IBT). The IBT showed a higher specificity than the CFT (Regalla et al., 2000) and it has been used, since January 1998, as a confirmatory test in CFT positive animals. The IBT specificity lies in the identification of a IgG core profile of 5 specific immunodominant antigenic bands of M. mycoides subspecies mycoides SC, with apparent molecular weights of 110, 98, 95, 62/60 and 48 kDa (Regalla et al., 2000). This profile is confirmatory of the individual infection. Between 1998 and 2002, a total of 20,588 CFT sero-reacting sera were examined by IBT and a total of 64 IgG immunoblot specific profiles were identified (Table 1). The last specific profile was detected in 2000 with no further confirmation of infection after herd slaughter.

The on-going serial testing (CF test and IBT), within the framework of Portuguese eradication programmes, has allowed the re-arrangement of the regions’ division of the territory. Since 1998, all the procedures applied before initiating the eradication programme conformed to a more stringent serological surveillance (CFT and IBT on positive CFT sera) allowing an accurate assessment of the CBPP sanitary situation in the Portuguese territory and provide the assurance of disease eradication.

References

Bashiruddin, J. B., Taylor, T. K. and Gould, A. R. (1994). A PCR-based test for the specific identification of Mycoplasma mycoides subspecies mycoides SC. Journal Veterinary Diagnostic Investigation 6, 428-434.

Regalla, J. (1984). Epidemiological aspects of contagious bovine pleuropneumonia in Portugal. Repositório de Trabalhos do Laboratório Nacional de Investigação Veterinária, XVI, 13-18

Regalla, J. (1995). La réaction de fixation du complément pour le diagnostic sérologique de la péripneumonie contagieuse bovine: application et interprétation des résultats. Revue Scientifique et Technique, 14 (3), 631-644.

Regalla, J., Caporale, V., Giovannini A., Santini, F., Martel, J. L. and Penha Gonçalves, A. (1996). Manifestation and epidemiology of contagious bovine pleuropneumonia in Europe. Revue Scientifique et Technique, 15 (4), 1309-1329.

Regalla, J., Gonçalves, R., Niza Ribeiro, J., Duarte, L., Nicholas, R., Bashiruddin, J., De Santis, P., Garrido Abellan, F. and Penha Gonçalves, A. (2000). Development of immunoblotting as a diagnostic tool for contagious bovine pleuropneumonia. In COST 826 Agriculture and biotechnology “Mycoplasmas of ruminants: pathogenicity, diagnostics, epidemiology and molecular genetics”, (Ed. D. Bergonier, X. Berthelot, and J. Frey), Rep. No. EUR 19245 EN, European Commission, Luxembourg, 109-112.

Table 1. Results of Complement Fixation test and immunoblotting test serial testing performed between 1998 and 2002, within the frameworks of Portuguese eradication programmes (Direcção Geral de Veterinária - Ministério da Agricultura e Pescas, Portugal)

CFT - Complement Fixation Test; IBT - Immunoblotting Test; CFt - Total number of CF tested