Fernando Boinas

The persistence of African swine fever virus in field-infected Ornithodoros erraticus during the ASF endemic period in Portugal.

African swine fever (ASF) is an important disease of pigs and outbreaks of ASF have occurred in Europe on multiple occasions. To explore the period for which the European soft tick species Ornithodoros erraticus (Acari: Argasidae) is able to act as a reservoir of African swine fever virus (ASFV) after infected hosts are removed, we collected specimens from farms in the provinces of Alentejo and Algarve in Portugal during the endemic period and tested them subsequently using cell culture and experimental infection. We show that ticks from previously infected farms may contain infectious virus for at least five years and three months after the removal of infectious hosts. Furthermore, in two cases infectious virus was successfully isolated from ticks on restocked farms that had not yet suffered a re-emergence of disease. Experimental transmission to pigs was demonstrated in batches tested up to 380 days after an outbreak. These results clarify the epidemiological role of O. erraticus ticks in the persistence of ASFV in the field, provide additional evidence to support its role in the re-emergence of a sporadic outbreak of ASF in Portugal in 1999 and suggest that the current quarantine legislation and restocking advice when these ticks are present on the pig farm premises is appropriate.

Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus

Summary.A nested PCR assay, with an internal control, was developed to detect African swine fever virus (ASFV) DNA in Ornithodoros erraticus. The assay revealed a better analytical sensitivity than virus isolation and the OIE PCR protocol. All ticks collected from the field, which were positive by virus isolation, were also positive by PCR. Viral DNA was detected in a further 19 out of 60 ticks from which no virus was isolated. Our results show that this assay is reliable and can easily be used to screen large tick populations collected in the field for the presence of ASFV.

Eradication of bovine brucellosis in the Azores, Portugal - Outcome of a 5-year programme (2002-2007) based on test-and-slaughter and RB51 vaccination.

Bovine brucellosis is an important contagious disease that can cause abortions and infertility in cattle, and can be transmitted to humans. Despite having an eradication programme in place since 1994, in 2000 the situation of bovine brucellosis due to Brucella abortus was not significantly improving in 3 of the 9 islands (Terceira, S. Miguel and S. Jorge) of the archipelago of Azores, an autonomous region of Portugal. Farming on these islands, particularly dairy, is extensive. Therefore, the use of RB51 vaccine, which does not induce antibodies detectable with routine brucellosis diagnostic tests, was implemented. This article reports the results of an eradication programme based on RB51 mass vaccination combined with test-and-slaughter, which was implemented in the Azores during the 2002-2007 period. During the first round of vaccination, both adult cows and heifers were vaccinated. Subsequently, only replacement stock aged 4-12 months, were immunized with RB51.The test-and-slaughter policy based on bulk milk ring test (MRT) and serological surveillance was maintained. During this period, the average brucellosis herd incidence, herd prevalence and individual prevalence decreased 69.26%, 39.26% and 75.41% respectively for the three above-mentioned islands. However, disease reduction approaching eradication was obtained only on the island of Terceira, where a high level of vaccine coverage was rapidly reached and regularly maintained together with strict application of a test-and-slaughter programme. This work shows that the RB51 vaccine could be a useful tool for eradicating bovine brucellosis in well-controlled epidemiological units provided that there is mass vaccine coverage for a sufficiently long period of time and it is combined with an appropriate test-and-slaughter programme.

African horse sickness in Portugal: a successful eradication programme.

African horse sickness (AHS) was diagnosed for the first time in southern Portugal in autumn 1989, following outbreaks in Spain. AHS virus presence was confirmed by virus isolation and serotyping. An eradication campaign with four sanitary zones was set up by Central Veterinary Services in close collaboration with private organizations. Vaccination began on 6 October. In February 1990, vaccination was extended to all Portuguese equines (170000 animals). There were 137 outbreaks on 104 farms: 206 of the equidae present died (16%) or were slaughtered (14%); 81.5% were horses, 10.7% were donkeys and 7.8% were mules. Clinical AHS occurred more frequently in horses than donkeys and mules. In the vaccinated population, 82 animals (62.2% horses and 37.8% mules and donkeys), died or were slaughtered due to suspected or confirmed AHS. One year after ending vaccination, December 1991, Portugal was declared free of AHS. Cost of eradication was US

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal.

1955513 (US

The CD2v protein enhances African swine fever virus replication in the tick vector, Ornithodoros erraticus

11.5/Portuguese equine).

The use of carbon dioxide insect traps for the collection of Ornithodoros erraticus on African swine fever-infected farms

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.

Borrelia hispanica in Ornithodoros erraticus, Portugal

The NH/P68 non-haemadsorbing (non-HAD) African swine fever virus (ASFV) isolate contains frameshift mutations in the EP402R and adjacent EP153R genes. These encode, respectively, the protein (CD2v) that is required for the haemadsorption (HAD) of swine erythrocytes to ASFV-infected cells and a C-type lectin protein. Two recombinant HAD viruses were constructed in this parental strain. In one of these the intact EP153R gene sequence was restored. Although restoration of the HAD phenotype did not increase virus virulence in pigs, a significant increase was observed in the number of pigs which developed viraemia. These HAD recombinant viruses replicated to titres approximately 1000-fold higher than the parental non-HAD isolate when membrane fed to Ornithodoros erraticus ticks. Inoculation of the non-HAD isolate across the gut wall increased viral replication to levels comparable to that of the HAD recombinant viruses. These results demonstrate a novel role for the CD2v protein in virus replication in ticks.

The medical and veterinary role of Ornithodoros erraticus complex ticks (Acari: Ixodida) on the Iberian Peninsula

Abstract The establishment of epidemiological studies of African swine fever involves the collection of large numbers of the soft tick, Ornithodoros erraticus, to assess the maintenance and spread of the disease in the semi-arid southern areas of Portugal. An on-farm monitoring system involving solid carbon dioxide trapping of ticks was used. This capture method proved to be both simple and effective when compared with manual collection. Both adult and stadial ticks were attracted by the traps making this method suitable for epidemiological surveillance studies of any disease which involves the Ornithodoros as a vector.

Use of a Bayesian network model to identify factors associated with the presence of the tick Ornithodoros erraticus on pig farms in southern Portugal

Tick-borne relapsing fever (TBRF) is a spirochetal infection caused by the genus Borrelia. The disease is distributed in the Old and New World with many different species reported. In Europe, TBRF is caused by B. hispanica transmitted to man by Ornithodoros erraticus, a soft tick usually found in old premises to shelter pig herds. In Portugal, the first human case of TBRF was reported in 1942 but since the beginning of the 1960s, the disease has rarely been described and seems to either have disappeared or have been undiagnosed. Therefore, in 2009 a survey was undertaken to evaluate the presence of the tick in this type of premises and to evaluate its role as a reservoir of Borrelia. The work was carried out where the ticks were previously reported in the Alentejo and Algarve regions. Of 63 pigpens surveyed, O. erraticus was collected from 19% (n = 12) of these pigpens using CO(2) traps. To evaluate potential Borrelia hosts, both pigs (n = 25) and small rodents (n = 10) inhabiting these pigpens were surveyed for Borrelia presence, by whole blood PCR and/or tissue culture, respectively. All results for pigs and rodents were negative for the presence of B. hispanica. PCR assays targeting the 16S rRNA gene and intergenic spacer region of Borrelia were used. Sequence analysis of the positive samples confirmed the presence of B. hispanica in 2.2% (n = 5) of ticks from a pigpen in Alentejo. These results confirm natural, but albeit low, persistence of this agent in Portugal.