Mosquito (Diptera: Culicidae) Fauna of a Zoological Park in an Urban Setting: Analysis of Culex pipiens s.l. and Their Biotypes.

Mosquito-borne diseases (MBDs) are important emerging diseases that affect humans and animals. Zoological parks can work as early warning systems for the occurrence of MBDs. In this study, we characterized the mosquito fauna captured inside Lisbon Zoo from May 2018 to November 2019. An average of 2.4 mosquitos per trap/night were captured. Five mosquito species potentially causing MBDs, including Culex pipiens biotypes, were found in the zoo. The sympatric occurrence of Culex pipiens biotypes represents a risk factor for the epizootic transmission of West Nile virus and Usutu virus. The mosquito occurrence followed the expected seasonality, with the maximum densities during summer months. However, mosquito activity was detected in winter months in low numbers. The minimum temperature and the relative humidity (RH) on the day of capture showed a positive effect on Culex pipiens abundance. Contrary, the RH the week before capture and the average precipitation the week of capture had a negative effect. No invasive species were identified, nor have flaviviruses been detected in the mosquitoes. The implementation of biosecurity measures regarding the hygiene of the premises and the strict control of all the animals entering the zoo can justify the low prevalence of mosquitoes and the absence of flavivirus-infected mosquitoes.

Targeted mutagenesis of the ß-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition.

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the ß-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

An Updated Review of Ornithodoros Ticks as Reservoirs of African Swine Fever in Sub-Saharan Africa and Madagascar.

This updated review provides an overview of the available information on Ornithodoros ticks as reservoirs and biological vectors of the ASF virus in Africa and Indian Ocean islands in order to update the current knowledge in this field, inclusive of an overview of available methods to investigate the presence of ticks in the natural environment and in domestic pig premises. In addition, it highlights the major areas of research that require attention in order to guide future investigations and fill knowledge gaps. The available information suggests that current knowledge is clearly insufficient to develop risk-based control and prevention strategies, which should be based on a sound understanding of genotype distribution and the potential for spillover from the source population. Studies on tick biology in the natural and domestic cycle, including genetics and systematics, represent another important knowledge gap. Considering the rapidly changing dynamics affecting the African continent (demographic growth, agricultural expansion, habitat transformation), anthropogenic factors influencing tick population distribution and ASF virus (ASFV) evolution in Africa are anticipated and have been recorded in southern Africa. This dynamic context, together with the current global trends of ASFV dissemination, highlights the need to prioritize further investigation on the acarological aspects linked with ASF ecology and evolution.

A DNA barcode reference library of Portuguese mosquitoes.

Mosquitoes are important biological vectors of pathogens and species identification in all life stages is the first step for effective monitoring and control of mosquito-borne diseases. Molecular methods for species identification have been developed over the last years to overcome the limitations of the taxonomic identification based on morphology. DNA barcoding, using a fragment of the mitochondrial cytochrome oxidase I (COI) gene, can be used for species identification but a reliable and comprehensive reference database of verified sequences is required. In this study, we aimed to generate a DNA barcode reference library for the identification of mosquito species from Portuguese mosquito fauna, including most relevant vector species. Mosquitoes captured under the National Vector Surveillance Program (REVIVE) were processed for DNA extraction, COI gene fragment amplification and sequencing. Nighty-eight barcode sequences were obtained, representing 26 species and 6 genera. Sequences were submitted to GenBank and BOLD and were used for validation of phenetic classification. Barcode Index Number (BIN) assignment and Automatic Barcode Gap Discovery (ABGD) were used and clustered COI sequences into twenty-five molecular operational taxonomic units (MOTUs). This is the first comprehensive study that combines morphological and molecular identification of most mosquito species present in Portugal aiming to offer a reliable framework for mosquito species identification.

Differential vector competence of Ornithodoros soft ticks for African swine fever virus: What if it involves more than just crossing organic barriers in ticks?

BACKGROUND: Several species of soft ticks in genus Ornithodoros are known vectors and reservoirs of African swine fever virus (ASFV). However, the underlying mechanisms of vector competence for ASFV across Ornithodoros species remain to be fully understood. To that end, this study compared ASFV replication and dissemination as well as virus vertical transmission to descendants between Ornithodoros moubata, O. erraticus, and O. verrucosus in relation to what is known about the ability of these soft tick species to transmit ASFV to pigs. To mimic the natural situation, a more realistic model was used where soft ticks were exposed to ASFV by allowing them to engorge on viremic pigs. METHODS: Ornithodoros moubata ticks were infected with the ASFV strains Liv13/33 (genotype I) or Georgia2007/1 (genotype II), O. erraticus with OurT88/1 (genotype I) or Georgia2007/1 (genotype II), and O. verrucosus with Ukr12/Zapo (genotype II), resulting in five different tick-virus pairs. Quantitative PCR (qPCR) assays targeting the VP72 ASFV gene was carried out over several months on crushed ticks to study viral replication kinetics. Viral titration assays were also carried out on crushed ticks 2 months post infection to confirm virus survival in soft ticks. Ticks were dissected. and DNA was individually extracted from the following organs to study ASFV dissemination: intestine, salivary glands, and reproductive organs. DNA extracts from each organ were tested by qPCR. Lastly, larval or first nymph-stage progeny emerging from hatching eggs were tested by qPCR to assess ASFV vertical transmission. RESULTS: Comparative analyses revealed higher rates of ASFV replication and dissemination in O. moubata infected with Liv13/33, while the opposite was observed for O. erraticus infected with Georgia2007/1 and for O. verrucosus with Ukr12/Zapo. Intermediate profiles were found for O. moubata infected with Georgia2007/1 and for O. erraticus with OurT88/1. Vertical transmission occurred efficiently in O. moubata infected with Liv13/33, and at very low rates in O. erraticus infected with OurT88/1. CONCLUSIONS: This study provides molecular data indicating that viral replication and dissemination in Ornithodoros ticks are major mechanisms underlying ASFV horizontal and vertical transmission. However, our results indicate that other determinants beyond viral replication also influence ASFV vector competence. Further research is required to fully understand this process in soft ticks.

Comparative vector competence of the Afrotropical soft tick Ornithodoros moubata and Palearctic species, O. erraticus and O. verrucosus, for African swine fever virus strains circulating in Eurasia.

African swine fever (ASF) is a lethal hemorrhagic disease in domestic pigs and wild suids caused by African swine fever virus (ASFV), which threatens the swine industry globally. In its native African enzootic foci, ASFV is naturally circulating between soft ticks of the genus Ornithodoros, especially in the O. moubata group, and wild reservoir suids, such as warthogs (Phacochoerus spp.) that are bitten by infected soft ticks inhabiting their burrows. While the ability of some Afrotropical soft ticks to transmit and maintain ASFV is well established, the vector status of Palearctic soft tick species for ASFV strains currently circulating in Eurasia remains largely unknown. For example, the Iberian soft tick O. erraticus is a known vector and reservoir of ASFV, but its ability to transmit different ASFV strains has not been assessed since ASF re-emerged in Europe in 2007. Little is known about vector competence for ASFV in other species, such as O. verrucosus, which occurs in southern parts of Eastern Europe, including Ukraine and parts of Russia, and in the Caucasus. Therefore, we conducted transmission trials with two Palearctic soft tick species, O. erraticus and O. verrucosus, and the Afrotropical species O. moubata. We tested the ability of ticks to transmit virulent ASFV strains, including one of direct African origin (Liv13/33), and three from Eurasia that had been involved in previous (OurT88/1), and the current epizooties (Georgia2007/1 and Ukr12/Zapo). Our experimental results showed that O. moubata was able to transmit the African and Eurasian ASFV strains, whereas O. erraticus and O. verrucosus failed to transmit the Eurasian ASFV strains. However, naïve pigs showed clinical signs of ASF when inoculated with homogenates of crushed O. erraticus and O. verrucosus ticks that fed on viraemic pigs, which proved the infectiousness of ASFV contained in the ticks. These results documented that O. erraticus and O. verrucosus are unlikely to be capable vectors of ASFV strains currently circulating in Eurasia. Additionally, the persistence of infection in soft ticks for several months reaffirms that the infectious status of a given tick species is only part of the data required to assess its vector competence for ASFV.

Geographical distribution of Culicoides (DIPTERA: CERATOPOGONIDAE) in mainland Portugal: Presence/absence modelling of vector and potential vector species.

Vector-borne diseases are not only accounted responsible for their burden on human health-care systems, but also known to cause economic constraints to livestock and animal production. Animals are affected directly by the transmitted pathogens and indirectly when animal movement is restricted. Distribution of such diseases depends on climatic and social factors, namely, environmental changes, globalization, trade and unplanned urbanization. Culicoides biting midges are responsible for the transmission of several pathogenic agents with relevant economic impact. Due to a fragmentary knowledge of their ecology, occurrence is difficult to predict consequently, limiting the control of these arthropod vectors. In order to understand the distribution of Culicoides species, in mainland Portugal, data collected during the National Entomologic Surveillance Program for Bluetongue disease (2005-2013), were used for statistical evaluation. Logistic regression analysis was preformed and prediction maps (per season) were obtained for vector and potentially vector species. The variables used at the present study were selected from WorldClim (two climatic variables) and CORINE databases (twenty-two land cover variables). This work points to an opposite distribution of C. imicola and species from the Obsoletus group within mainland Portugal. Such findings are evidenced in autumn, with the former appearing in Central and Southern regions. Although appearing northwards, on summer and autumn, C. newsteadi reveals a similar distribution to C. imicola. The species C. punctatus appears in all Portuguese territory throughout the year. Contrary, C. pulicaris is poorly caught in all areas of mainland Portugal, being paradoxical present near coastal areas and higher altitude regions.

Prevalence and factors associated with human brucellosis in livestock professionals.

OBJECTIVE: The objective of this study is to estimate the seroprevalence of human brucellosis in livestock professionals and analyze the factors associated with brucellosis focusing on sociodemographic variables and the variables of knowledge and practices related to the characteristics of the activities carried out in livestock. METHODS: This is a cross-sectional seroepidemiological study with a population of 131 workers of butchers, slaughter rooms, and slaughterhouse and 192 breeders sampled randomly in Namibe province, Angola. The data were obtained from the collection of blood and use of questionnaires. The laboratory tests used were rose bengal and slow agglutination. The questionnaire allowed us to collect sociodemographic information and, specifically on brucellosis, it incorporated questions about knowledge, attitudes, and behaviors of livestock professionals. In addition to the descriptive statistical approach, we used the Chi-square test of independence, Fisher's test, and logistic regression models, using a significance level of 10%. RESULTS: The general weighted prevalence of brucellosis was 15.6% (95%CI 13.61-17.50), being it 5.3% in workers and 16.7% (95%CI 11.39-21.93) in breeders. The statistical significance was observed between human seroprevalence and category (worker and breeder) (p < 0.001) and education level (p = 0.032), start of activity (p = 0079), and service location (p = 0.055). In a multivariate context, the positive factor associated with brucellosis in professionals was the professional category (OR = 3.54; 95%CI 1.57-8.30, related to breeders in relation to workers). CONCLUSIONS: Human brucellosis in livestock professionals is prevalent in Namibe province (15.6%), where the professional category was the most important factor. The seroprevalence levels detected are high when compared with those found in similar studies. OBJECTIVO: Estimar a seroprevalência da brucelose humana em profissionais da pecuária e analisar os factores associados à brucelose com foco em variáveis sociodemográficas, de conhecimento e práticas relativas às características das actividades desenvolvidas na pecuária. MÉTODOS: Estudo transversal seroepidemiológico em população de 131 trabalhadores de talhos, salas de abate e matadouro e 192 criadores amostrados aleatoriamente na província do Namibe, Angola. Os dados foram obtidos por meio de colheita de sangue e aplicação de questionário. Os testes laboratoriais utilizados foram o Rosa de Bengala e a aglutinação lenta em tubos. O questionário permitiu recolher informação sociodemográfica e, especificamente sobre a brucelose, incorporou questões sobre conhecimento, atitudes e comportamentos dos profissionais da pecuária. Além da abordagem estatística descritiva, foram utilizados os testes de Independência do Qui-quadrado, Fisher e modelos de regressão logística, utilizando um nível de significância de 10%. RESULTADOS: A prevalência geral ponderada da brucelose foi de 15.6% (IC95% 13.61-17.50), sendo 5.3% em trabalhadores e 16.7% (IC95% 11.39-21.93) em criadores. A significância estatística foi observada entre a seroprevalência humana e a categoria (trabalhador e criador) (p < 0.001) e o nível de instrução (p = 0.032), início de actividade (p = 0.079) e local de serviço (p = 0.055). Em um contexto multivariado, o factor positivamente associado à brucelose em profissionais foi a categoria profissional (OR = 3.54; IC95% 1.57-8.30, relativo aos criadores em relação a trabalhadores). CONCLUSÕES: A brucelose humana em profissionais da pecuária é prevalente na província do Namibe (15.6%), onde a categoria profissional foi o factor mais relevante. Os níveis de seroprevalência detectados são elevados se comparados com outros encontrados em estudos semelhantes.

Prevalence and factors associated with human brucellosis in livestock professionals / Prevalência e factores associados à brucelose humana em profissionais da pecuária

ABSTRACT OBJECTIVE The objective of this study is to estimate the seroprevalence of human brucellosis in livestock professionals and analyze the factors associated with brucellosis focusing on sociodemographic variables and the variables of knowledge and practices related to the characteristics of the activities carried out in livestock. METHODS This is a cross-sectional seroepidemiological study with a population of 131 workers of butchers, slaughter rooms, and slaughterhouse and 192 breeders sampled randomly in Namibe province, Angola. The data were obtained from the collection of blood and use of questionnaires. The laboratory tests used were rose bengal and slow agglutination. The questionnaire allowed us to collect sociodemographic information and, specifically on brucellosis, it incorporated questions about knowledge, attitudes, and behaviors of livestock professionals. In addition to the descriptive statistical approach, we used the Chi-square test of independence, Fisher’s test, and logistic regression models, using a significance level of 10%. RESULTS The general weighted prevalence of brucellosis was 15.6% (95%CI 13.61–17.50), being it 5.3% in workers and 16.7% (95%CI 11.39–21.93) in breeders. The statistical significance was observed between human seroprevalence and category (worker and breeder) (p < 0.001) and education level (p = 0.032), start of activity (p = 0079), and service location (p = 0.055). In a multivariate context, the positive factor associated with brucellosis in professionals was the professional category (OR = 3.54; 95%CI 1.57–8.30, related to breeders in relation to workers). CONCLUSIONS Human brucellosis in livestock professionals is prevalent in Namibe province (15.6%), where the professional category was the most important factor. The seroprevalence levels detected are high when compared with those found in similar studies.

RESUMO OBJECTIVO Estimar a seroprevalência da brucelose humana em profissionais da pecuária e analisar os factores associados à brucelose com foco em variáveis sociodemográficas, de conhecimento e práticas relativas às características das actividades desenvolvidas na pecuária. MÉTODOS Estudo transversal seroepidemiológico em população de 131 trabalhadores de talhos, salas de abate e matadouro e 192 criadores amostrados aleatoriamente na província do Namibe, Angola. Os dados foram obtidos por meio de colheita de sangue e aplicação de questionário. Os testes laboratoriais utilizados foram o Rosa de Bengala e a aglutinação lenta em tubos. O questionário permitiu recolher informação sociodemográfica e, especificamente sobre a brucelose, incorporou questões sobre conhecimento, atitudes e comportamentos dos profissionais da pecuária. Além da abordagem estatística descritiva, foram utilizados os testes de Independência do Qui-quadrado, Fisher e modelos de regressão logística, utilizando um nível de significância de 10%. RESULTADOS A prevalência geral ponderada da brucelose foi de 15.6% (IC95% 13.61–17.50), sendo 5.3% em trabalhadores e 16.7% (IC95% 11.39–21.93) em criadores. A significância estatística foi observada entre a seroprevalência humana e a categoria (trabalhador e criador) (p < 0.001) e o nível de instrução (p = 0.032), início de actividade (p = 0.079) e local de serviço (p = 0.055). Em um contexto multivariado, o factor positivamente associado à brucelose em profissionais foi a categoria profissional (OR = 3.54; IC95% 1.57–8.30, relativo aos criadores em relação a trabalhadores). CONCLUSÕES A brucelose humana em profissionais da pecuária é prevalente na província do Namibe (15.6%), onde a categoria profissional foi o factor mais relevante. Os níveis de seroprevalência detectados são elevados se comparados com outros encontrados em estudos semelhantes.

Experimental Infection of Ornithodoros erraticus sensu stricto with Two Portuguese African Swine Fever Virus Strains. Study of Factors Involved in the Dynamics of Infection in Ticks.

African swine fever (ASF) is a frequently devastating hemorrhagic disease of domestic pigs and wild boar and Ornithodoros erraticus sensu stricto argasid ticks are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe. Recently this disease emerged in Eastern Europe and Russian Federation, showing a huge potential for a rapid spread between countries. There is some risk of re-emergence of ASF in the countries where these ticks exist, that can contribute for the persistence of infection and compromise control measures. In this study we aimed to identify factors that determine the probability of infection and its dynamics in the tick vector Ornithodoros erraticus sensu stricto, with two Portuguese strains of ASFV. Our results suggest that these ticks have a high likelihood of excreting the two haemadsorbing ASF viruses of different host origins and that, in field surveys, the analysis of adults and 5th nymphal stage can provide the best chance of detecting virus infection. The results also indicate that infection of pigs with highly virulent ASF viruses will promote higher rates of infection and a higher likelihood for virus excretion by ticks. Nevertheless, there is also a risk, although lower, that ticks can become infected on pigs that have overcome the acute phase of infection, which was simulated in our study by membrane feeding ticks with low titres of virus. We believe these results can be valuable in designing and interpreting the results of ASF control programmes, and future work can also be undertaken as our dataset is released under open access, to perform studies in risk assessment for ASFV persistence in a region where O. erraticus sensu stricto ticks are present.

Spatial and temporal distribution of Culicoides species in mainland Portugal (2005-2010). Results of the Portuguese Entomological Surveillance Programme.

Bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes an infectious, non-contagious disease of ruminants. It has been rapidly emerging in southern Europe since 1998. In mainland Portugal, strains of BTV belonging to three serotypes have been detected: BTV-10 (1956-1960), BTV-4 (2004-2006 and 2013) and BTV-1 (2007-2012). This paper describes the design, implementation and results of the Entomological Surveillance Programme covering mainland Portugal, between 2005 and 2010, including 5,650 caches. Culicoides imicola Kieffer was mostly found in central and southern regions of Portugal, although it was sporadically detected in northern latitudes. Its peak activity occurred in the autumn and it was active during the winter months in limited areas of the country. Obsoletus group was present at the highest densities in the north although they were found throughout the country in substantial numbers. Culicoides activity occurred all year round but peaked in the spring. A generalized linear mixed model was developed for the analysis of the environmental factors associated with activity of the species of Culicoides suspected vectors of BTV in the country. For C. imicola Kieffer, the most important variables were month, diurnal temperature range (DTR), the number of frost days (FRS) and median monthly temperature (TMP). For the Obsoletus group, the most important factors were month, diurnal temperature range (DTR), and linear and quadratic terms for median monthly temperature (TMP). The results reported can improve our understanding of climatic factors in Culicoides activity influencing their distribution and seasonal pattern.

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

Argasid ticks of the Ornithodoros erraticus complex are associated with traditional pig-farming practices on the Iberian Peninsula and are also found elsewhere in North Africa, West Africa, and western Asia. The ticks associated with pig farming on the Iberian Peninsula are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe, and their ecology makes them an extremely effective reservoir of both ASFV and the Borrelia species which cause tick-borne relapsing fever (TBRF) in humans. The recent reappearance of ASFV in the European Union, coupled with evidence that Portuguese tick populations continue to harbor Borrelia despite a lack of confirmed human infections, suggest that these populations merit closer attention. In Portugal, a series of surveys over the last twenty-five years indicates that the number of farm sites with tick infestations has declined and suggest that populations are sensitive to changes in farm management, particularly the use of modern pig housing. Various technologies have been suggested for the control of farm-associated Ornithodoros ticks and related species but, in our opinion, farm management changes are still the most effective strategy for population control. Furthermore, we suggest that this species could probably be eradicated from Iberian pig farms.

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

The soft tick Ornithodoros erraticus occurs on pig farms in southern Portugal and Spain and transmits several important pathogens of humans and livestock. Its distribution is patchy and the determinants of its distribution are uncertain. Here, we use a Bayesian network model to explore possible associations between climate, farm management and the presence of O. erraticus. The resulting network confirms previous suggestions that the presence of O. erraticus is more likely in traditionally constructed pig housing, and indicates that carbon dioxide traps are highly effective for the detection of O. erraticus. Our approach also picked up several other intuitively reasonable relationships, including that traditional housing was more likely to be in poor condition and more likely to be out of use, and that buildings which were in use to house pigs were also less likely to be derelict. Neither temperature nor precipitation had any direct effect on the probability of the presence of O. erraticus, suggesting that the distribution of the species is primarily driven by farm management factors.

First report of 13 species of Culicoides (Diptera: Ceratopogonidae) in mainland Portugal and Azores by morphological and molecular characterization.

The genus Culicoides (Diptera: Ceratopogonidae) contains important vectors of animal and human diseases, including bluetongue, African horse sickness and filariosis. A major outbreak of bluetongue occurred in mainland Portugal in 2004, forty eight years after the last recorded case. A national Entomological Surveillance Plan was initiated in mainland Portugal, Azores and the Madeira archipelagos in 2005 in order to better understand the disease and facilitate policy decisions. During the survey, the most prevalent Culicoides species in mainland Portugal was C. imicola (75.3%) and species belonging to the Obsoletus group (6.5%). The latter were the most prevalent in Azores archipelago, accounting for 96.7% of the total species identified. The Obsoletus group was further characterized by multiplex Polymerase Chain Reaction to species level showing that only two species of this group were present: C. obsoletus sensu strictu (69.6%) and C. scoticus (30.4%). Nine species of Culicoides were detected for the first time in mainland Portugal: C. alazanicus, C. bahrainensis, C. deltus, C. lupicaris, C. picturatus, C. santonicus, C. semimaculatus, C. simulator and C. subfagineus. In the Azores, C. newsteadi and C. circumscriptus were identified for the first time from some islands, and bluetongue vectors belonging to the Obsoletus group (C. obsoletus and C. scoticus) were found to be widespread.

Detection of Rickettsia conorii strains in Portuguese dogs (Canis familiaris).

This study reports a serological and molecular survey on the spotted fever group Rickettsia found in a group of 51 dogs with suspected tick-borne illness from the south of Portugal. Additionally, a prevalence of IgG antibodies to R. conorii was also estimated in a group of 400 healthy dogs from the same region. In the group of healthy dogs, the immunofluorescence test revealed that 154 (38.5%) of the 400 dogs had IgG antibodies reactive with R. conorii. The highest proportion of dogs (45%) with R. conorii antibodies was found in blood samples collected from October to December. Among the group suspected with tick-borne illness, 35 (62%) dogs showed to be seroreactive (IgG≥128) for antibodies against R. conorii, and the analysis of PCR-positive amplicons revealed that 5 dogs were infected with R. conorii Malish and 2 dogs were infected with R. conorii Israeli tick typhus strain. There was a higher prevalence of antibodies to R. conorii in the group of sick dogs, and the detection of R. conorii DNA in blood samples from this group points to their potential role as a reservoir and sentinel host helping to evaluate and characterize the distribution of circulating rickettsial strains.

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.

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

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.

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

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.

Kinetics of African swine fever virus infection in Ornithodoros erraticus ticks.

The kinetics of African swine fever virus (ASFV) infection in Ornithodoros erraticus ticks were investigated in specimens collected in the field at different times following an outbreak of the disease in Portugal in 1999 and in ticks infected experimentally with a virus isolated from a tick collected during this outbreak. In ticks collected from the field, initial screening for ASFV was carried out by PCR, followed by attempts to isolate the virus in macrophage cultures. Considering total numbers of ticks tested independently of developmental stages, ASFV DNA was detected in 42.3, 26.4 and 22.4% of specimens collected at weeks 0, 32 and 63 following the outbreak, respectively. Although virus was not isolated from most of these ticks, the proportion of isolations from large nymphs and adults increased between weeks 0 and 32 from 2 to 9 % and from 5 to 11.5%, respectively. These results, together with the higher virus titres at week 32, suggest that virus replication occurred. In contrast, virus isolations from small nymphs decreased over this period, from 5 to 1.3%. At week 63, infection rates decreased for all stages. Experimental infections showed the occurrence of virus replication within 4 weeks post-feeding and maintenance of high titres in almost 100% of ticks until 20 weeks post-infection. At weeks 41 and 61, a drop in virus titres and infection rates was observed. Relevant to the understanding of African swine fever epidemiology, our results show that ASFV replicates and persists in O. erraticus, but a viral clearance occurs at later times in both natural and experimental infections.