Simultaneous Determination of Neutron-Induced Fission and Radiative Capture Cross Sections from Decay Probabilities Obtained with a Surrogate Reaction.

Reliable neutron-induced-reaction cross sections of unstable nuclei are essential for nuclear astrophysics and applications but their direct measurement is often impossible. The surrogate-reaction method is one of the most promising alternatives to access these cross sections. In this work, we successfully applied the surrogate-reaction method to infer for the first time both the neutron-induced fission and radiative capture cross sections of ^{239}Pu in a consistent manner from a single measurement. This was achieved by combining simultaneously measured fission and γ-emission probabilities for the ^{240}Pu(^{4}He,^{4}He^{'}) surrogate reaction with a calculation of the angular-momentum and parity distributions populated in this reaction. While other experiments measure the probabilities for some selected γ-ray transitions, we measure the γ-emission probability. This enlarges the applicability of the surrogate-reaction method.

Increased prevalence of Rickettsia aeschlimannii in Castilla y León, Spain.

We report the increased prevalence recorded in recent years of Rickettsia aeschlimannii in Hyalomma marginatum marginatum ticks removed from human subjects in Castilla y León (NW, Spain). Additionally, a simultaneous infection with R. aeschlimannii and Anaplasma phagocytophilum in two H. marginatum marginatum ticks is reported for the first time.

Acaricidal activity of fluralaner against Ornithodoros moubata and Ornithodoros erraticus argasid ticks evaluated through in vitro feeding.

Ornithodoros erraticus and Ornithodoros moubata are argasid tick vectors that transmit severe diseases such as African swine fever and human relapsing fever. Elimination of the synanthropic populations of these vectors would facilitate the control of these diseases. Fluralaner is a novel isoxazoline that selectively blocks the GABA- and glutamate-gated channels, providing potent insecticidal and acaricidal activity. The aim of the current study was to provide quantitative data on the susceptibility of males, females and third nymphal instar of O. erraticus and O. moubata to fluralaner through in vitro feeding exposure. Fluralaner activity against these developmental stages and species was assessed by feeding the ticks on ovine blood medicated with decreasing fluralaner concentrations between 1 and 10-8µg/mL. Tick mortality was measured at 4, 24 and 48h and 1, 2 and 3 weeks post-feeding. Tests included solvent-treated and untreated blood controls. Fluralaner was extremely active against O. erraticus, with mean lethal concentrations 50 (LC50) and 95 (LC95) of 2.0×10-8 and 5.4×10-8µg/mL, respectively. Fluralaner was also highly active against O. moubata, showing a mean LC50 of 1.5×10-6µg/mL and a mean LC95 of 1.8×10-3µg/mL. In the latter species, the most susceptible life stages were the females (LC95 1.4×10-4µg/mL). Fluralaner demonstrated potent acaricidal activity against all developmental stages of O. erraticus and O. moubata tested, in the first 48h after in vitro feeding. Therefore, fluralaner has the potential to provide very high acaricidal efficacy to multiple argasid tick species via feeding exposure and could be included as an acaricidal agent in integrated programmes for the control of argasid tick vectors and argasid tick-borne diseases.

Serological Surveillance and Direct Field Searching Reaffirm the Absence of Ornithodoros Erraticus Ticks Role in African Swine Fever Cycle in Sardinia.

African swine fever (ASF), one of the most important diseases of swine, has been endemic in the Italian island of Sardinia for more than 35 years. During these decades, several strategies and eradication efforts have been implemented in the island with limited success. Strong climatic and ecological similarities exist between Sardinia and one area of the Iberian Peninsula where Ornithodoros erraticus ticks were involved in the persistence of ASF from 1960 to 1995. This fact leads to the hypothesis that, potentially, Ornithodoros ticks could be also involved in the ASF cycle in Sardinia, thus accounting for some of the reoccurring ASF outbreaks in this island. Initial efforts aimed at detection of Ornithodoros ticks in Sardinia were performed during the 1970s/1980s with no positive results. Accordingly, the absence of Ornithodoros ticks in Sardinia has been generally accepted. However, since a new and reinforced ASF eradication programme has been recently launched in Sardinia, it is essential to clarify the presence and role of these soft ticks in the epizootiology of ASF in this island. For that purpose, 1767 porcine serum samples collected from all around the island (1261 from domestic and 506 from wild boar) were analysed by ELISA for antibodies to salivary antigens of Ornithodoros erraticus. In addition, Ornithodoros ticks were directly searched in a number of pig premises that have suitable habitats for these ticks and were located in areas repeatedly affected by ASF. Only one serum sample resulted positive in the serological assay, and no Ornithodoros ticks were collected in none of the premises. These results indicate that these soft tick species are not involved in the epizootic cycle of ASF in Sardinia and highlight the importance of controlling other risk factors still present in the island for effectively eradicate the disease.

Investigation into the Epidemiology of African Swine Fever Virus at the Wildlife - Domestic Interface of the Gorongosa National Park, Central Mozambique.

An epidemiological study of African swine fever (ASF) was conducted between March 2006 and September 2007 in a rural area adjacent to the Gorongosa National park (GNP) located in the Central Mozambique. Domestic pigs and warthogs were sampled to determine the prevalence of antibodies against ASF virus and the salivary antigens of Ornithodoros spp. ticks, while ticks collected from pig pens were tested for the presence of ASFV. In addition, 310 framers were interviewed to gain a better understanding of the pig value chain and potential practices that could impact on the spread of the virus. The sero-prevalence to ASFV was 12.6% on farms and 9.1% in pigs, while it reached 75% in warthogs. Approximately 33% of pigs and 78% of warthogs showed antibodies against salivary antigens of ticks. The differences in sero-prevalence between farms close to the GNP, where there is greater chance for the sylvatic cycle to cause outbreaks, and farms located in the rest of the district, where pig to pig transmission is more likely to occur, were marginally significant. Ornithodoros spp. ticks were found in only 2 of 20 pig pens outside the GNP, and both pens had ticks testing positive for ASFV DNA. Interviews carried out among farmers indicated that biosecurity measures were mostly absent. Herd sizes were small with pigs kept in a free-ranging husbandry system (65%). Only 1.6% of farmers slaughtered on their premises, but 51% acknowledged allowing visitors into their farms to purchase pigs. ASF outbreaks seemed to have a severe economic impact with nearly 36% of farmers ceasing pig farming for at least 1 year after a suspected ASF outbreak. This study provides the first evidence of the existence of a sylvatic cycle in Mozambique and confirms the presence of a permanent source of virus for the domestic pig value chain.

TSGP4 from Ornithodoros moubata: molecular cloning, phylogenetic analysis and vaccine efficacy of a new member of the lipocalin clade of cysteinyl leukotriene scavengers.

Recently obtained evidence indicated that an orthologue of the O. savignyi TSGP4 salivary lipocalin was present in the saliva of O. moubata. TSGP4 is known to act as a cysteinyl leukotrienes scavenger helping in the prevention of inflammation and oedema at the tick bite site. Since this function seems to be crucial for successful tick feeding, the novel O. moubata TSGP4 turned into a potential vaccine target. The purposes of the current work were: (i) to clone and characterize the O. moubata TSGP4 and, (ii) to produce it as recombinant to evaluate its protective efficacy as vaccine antigen. The results of these experiments indicated that the O. moubata TSGP4 shows high sequence and structural identity with the O. savignyi orthologue suggesting identical function in the physiology of the tick-host relationship. The mature native TSGP4 is not immunogenic when it is inoculated to host with tick saliva during feeding, but host vaccination with the recombinant protein TSGP4 in Freund's adjuvants induced strong humoral immune responses that recognized both the recombinant and native TSGP4 and protected the host with a 14.1% efficacy. So, the O. moubata TSGP4 can be considered a silent salivary antigen; however, in the light of the current results, its inclusion in the current repertory of protective antigens to be targeted by anti-tick vaccines could be controversial.

A study of the vaccinal value of various extracts of concealed antigens and salivary gland extracts against Ornithodoros erraticus and Ornithodoros moubata.

On pig farms, elimination of the argasid ticks acting as reservoirs and vectors for African swine fever greatly favours the eradication of this disease. The elimination of Ornithodoros erraticus involves many problems, most of which could be easily solved by the development of an anti-O. erraticus vaccine. With a view to developing this vaccine, we have tested the protective value of the immune response induced in swine by seven 'concealed' antigens and one soluble salivary gland extract. The latter extract was also prepared from Ornithodoros moubata specimens and tested against this tick. Our results indicate that the immune response elicited by the concealed antigens has no protective value against O. erraticus. The immune response induced by the salivary gland extracts against adults of O. erraticus and O. moubata was apparent in a reduced ingestion of blood (40-60%; P < 0.01) (except in males of O. erraticus) and in a significant decrease (40-60%; P < 0.01) in fecundity in 100% of the females of both species. The good results obtained with salivary antigens, which in situations of natural contact have no protective value, are attributed to the fact that when these antigens are injected with adjuvants, the immune system recognizes certain salivary components (probably those which enable the parasite to feed) which it does not recognize under natural conditions of exposure.

Detection of pig farms with Ornithodoros erraticus by pig serology. Elimination of non-specific reactions by carbohydrate epitopes of salivary antigens.

Ornithodoros erraticus is the European vector of African Swine Fever. It is therefore essential to know on which pig farms the tick is present in order to prevent contact with swine. Currently, studies are being made to ascertain this through the detection of anti-O. erraticus antibodies in the sera of swine, using three extracts from the salivary glands of the parasite (SGE): a complete extract (SGE-1), a soluble antigens extract (SGE-2), and a tissue antigens extract (SGE-3). The results of the present work show that SGE-2 gives the best differentiation between swine bitten by O. erraticus and unbitten swine in the enzyme-linked immunosorbent assay (ELISA). Using this extract, an optical density (OD) five-fold higher than the basal OD indicates that the pigs carry anti-O. erraticus antibodies. A serological study carried out in Salamanca with 8083 sera from 1756 pig farms revealed the presence of the parasite on 135 farms. However, during this study we noticed that some sera of unbitten animals gave false-positive reactions. Western blot analysis of SGE-2 of these false-positive sera demonstrated the same bands (except for two) as the real anti-O. erraticus sera. We observed, in ELISA and Western blot analysis, that such false-positive sera only recognised carbohydrate epitopes on SGE-2. This reactivity disappeared on deglycosylated SGE-2 (SGE-2-P). Therefore, SGE-2-P is the antigen that confers the greatest specificity to serology. In this study it was also observed that the low levels of anti-O. erraticus antibodies found in some cases may be because the swine were bitten some months previously on a different farm or that the current farm harboured only a few specimens of O. erraticus, so pig-tick contact is unlikely and hence the pigs either only develop a primary response or the time between contacts is very long and the levels of antibodies fall. Since pigs could be bitten on a different farm, the presence of low levels of anti-O. erraticus antibodies in pig sera do not necessarily indicate the presence of the tick on the farm where sampling was carried out.

Purification, N-terminal sequencing and diagnostic value of the major antigens of Ornithodoros erraticus and O. moubata.

To enhance the specificity and sensitivity of serological detection of swine exposed to Ornithodoros erraticus or O. moubata, we purified the 158, 186, 215 and 260 kDa antigens from the former species and the designated (owing to their MW and charge) 19C, 17A, 20A1 and 20A2 antigens of the latter by HPLC and gel electroelution methods. All the O. erraticus antigens share epitopes and are difficult to purify individually by reverse phase and ion-exchange chromatography due to their molecular similarity. Tested individually by ELISA, all of them give the same optical densities (OD) with anti-O. erraticus sera, and these ODs are always lower with anti-immature than with anti-adult sera. Although immature and adult specimens have the same antigens, immature forms induce more anti-carbohydrate antibodies than adults. This is the reason for the lower ODs of the anti-immature sera against purified antigens, since these latter antigens essentially react with anti-peptide antibodies (hence, increasing the specificity and sensitivity of the serology). The N-terminus of the 260 kDa antigen shows 80-90% similarity with the hemoglobin alpha-chain of many mammals. The antigens of O. moubata are proteins that are very different from one another and are, therefore, readily purified by ion exchange chromatography. The 20A1 antigen appears to be the most immunogenic and is recognized equally by anti-immature and anti-adult sera. This antigen does not give false positive reactions with the negative control sera analyzed and its N-terminus region shares 46.2% homology with the alpha-chain of the C3 component of rabbit complement.

Relationships between the defensive systems of Iberian-breed swine and the European vector of African swine fever, Ornithodoros erraticus.

To discover whether the immune system of Iberian-breed pigs exerts any adverse action on Ornithodoros erraticus, 3 pairs of pigs were subjected to a weekly infestation over 12 wk with 1,000 larvae, 500 nymphs-1, or 200 adults. Each pair was bitten by only 1 developmental stage. Batches of parasites identical to the foregoing ones were fed weekly on control swine. In none of the 10 parameters studied for each of the batches fed weekly was any significant difference found that could be attributed to the state of sensitization of the animals in which, in a previous study, the presence of high titers of anti-O. erraticus antibodies was found. It was observed that the possible pruritus due to immediate hypersensitivity reactions, which in the test animals appeared after the third week, had no protective value in the natural milieu. In view of the inability of the swine to exert any control over the soft ticks, it is concluded that the size of their populations in the pig pens and their composition according to the developmental stage are factors that depend exclusively on the opportunities that swine breeders offer such populations to feed on the animals.

Molecular detection of Ehrlichia phagocytophila genogroup organisms in larvae of Neotrombicula autumnalis (Acari: Trombiculidae) captured in Spain.

Twenty unfed larvae of Neotrombicula autumnalis (Acari: Trombiculidae) collected on vegetation in the north of Spain were examined by polymerase chain reaction for Borrelia burgdorferi (s.l.). rickettsiae, and the Ehrlichia phagocytophila genogroup. At least 10% of the larvae were found to contain granulocytic ehrlichiae. Because the larvae were unfed, they would necessarily have inherited the bacteria through a transovarian transmission pathway.

Antigens of interest for the diagnosis of parasitism in pigs by Ornithodoros erraticus and Ornithodoros moubata.

We show by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot that the composition of the soluble extracts of salivary glands (SGE-2) of Ornithodoros erraticus and Ornithodoros moubata is similar to that of the saliva (pilocarpine-induced), and that the extracts are a valid source of antigens for the detection of anti-argasid antibodies. It is also shown that the SGE-2s do not vary qualitatively with the developmental stage, physiological status, or sex of the ticks. The antigenic components (at least in O. erraticus) are released into the SGE-2 by the action of enzymes that can be inhibited by phenylmethane sulfonyl fluoride plus EDTA. Most of the components of the SGE-2, except the antigenic ones, are strongly glycosylated. Accordingly, the deglycosylation of the SGE-2s does not affect the recognition of antigenic components by anti-tick sera. In both species, the major components of the SGE-2s or the saliva are not recognized by the corresponding antisera. These nonimmunogenic components could have vaccinal value but not diagnostic interest. Finally, it is shown that the antigens of O. erraticus and O. moubata do not cross-react with one another and that those of the first species are more antigenic than those of the second.

Relationship between the persistence of African swine fever and the distribution of Ornithodoros erraticus in the province of Salamanca, Spain.

For some time it has been known that the tick Ornithodoros erraticus might be one of the causes of the persistence of African swine fever in the Iberian Peninsula since its introduction in 1960. In the province of Salamanca serological methods have been used to study the relationship between the presence of the tick in different townships and the outbreaks of African swine fever in these townships between 1987 and 1992. The results showed that there was a statistically significant association between the presence of the parasite and the persistence of African swine fever. In townships without O erraticus traditional methods of control are in most cases enough to avoid new outbreaks of the disease, but in areas with O erraticus the traditional methods should be reinforced by other methods for preventing contact between pigs and the parasite.

Distribution and biology of Ornithodoros erraticus in parts of Spain affected by African swine fever.

Ornithodoros erraticus was found in 30.7 per cent, 35.0 per cent and 71.0 per cent of the pig-pens sampled in the provinces of Salamanca, Badajoz and Huelva in which African swine fever is a problem in the rearing of Iberian pigs. Between 38 and 65 per cent of the pig-pens in these areas are now abandoned and their populations of O erraticus are extinct or becoming so because they can no longer feed on pigs, which in Spain are their main hosts. The abandonment of pig-pens has resulted in the elimination of most soft ticks infected with the virus of African swine fever, and means that the distribution of ticks is now irregular and focal. Another factor affecting their distribution is the kind of soil on which the pig-pens are located. In abandoned pig-pens, the adults and large nymphs survive for about five years or longer when animals occasionally enter them. Hungry tick populations may transmit African swine fever when feeding in winter, whereas the populations that have continuous access to pigs do not feed until the pig-pens reach a temperature of 13 to 15 degrees C. In the latter populations, each stage exhibits a single annual peak of activity, which implies that the development from larva to adult takes two to three years. Pigs may die as a result of the bites, but on no occasion were 100 per cent of the fasting ticks seen to feed, even though they had the opportunity of doing so. This may hinder the eradication of this soft tick from infested pig-pens.

Review of the sylvatic cycle of African swine fever in sub-Saharan Africa and the Indian ocean.

African swine fever (ASF) is a major limiting factor for pig production in most of the countries in Sub-Saharan Africa and the Indian Ocean. In the absence of vaccine, a good understanding of the ecology and epidemiology of the disease is fundamental to implement effective control measures. In selected countries of Southern and East Africa, the association between Ornithodoros moubata ticks and warthogs has been described in detail in the literature. However, for many other countries in the region, information related to the sylvatic cycle is lacking or incomplete. In West African countries, for instance, the role of wild pigs in the epidemiology of ASF has never been demonstrated and the existence and potential impact of a sylvatic cycle involving an association between soft ticks and warthogs is questionable. In other countries, other wild pig species such as the bushpigs (Potamochoerus spp.) can also be asymptomatically infected by the virus but their role in the epidemiology of the disease is unclear and might differ according to geographic regions. In addition, the methods and techniques required to study the role of wild hosts in ASF virus (ASFV) epidemiology and ecology are very specific and differ from the more traditional methods to study domestic pigs or other tick species. The aim of this review is (i) to provide a descriptive list of the methodologies implemented to study the role of wild hosts in African swine fever, (ii) to compile the available knowledge about the sylvatic cycle of ASFV in different regions of Sub-Saharan Africa and the Indian Ocean in addition to the one that has been described for East and Southern Africa, and (iii) to discuss current methodologies and available knowledge in order to identify new orientations for further field and experimental surveys.

Assessment of interactions between African swine fever virus, bushpigs (Potamochoerus larvatus), Ornithodoros ticks and domestic pigs in north-western Madagascar.

Since its introduction in Madagascar in 1998, African swine fever (ASF) has severely affected national pig production and persists as a common disease in that country. Two of its natural hosts in the African continent, the bushpig (Potamochoerus larvatus) and tick vectors of the Ornithodoros moubata complex, are reported in west and central regions of the island. However, their role in the maintenance and transmission of the virus has been insufficiently studied. In this work, we tried to assess their potential role in the epidemiology of the disease in Madagascar, by assessing the levels of interaction between (i) ASF virus (ASFV) and bushpigs and (ii) between soft ticks and domestic and wild suids in north-western Madagascar. Twenty-seven sera and 35 tissue samples from bushpigs were collected and analysed for the presence of anti-ASF antibodies and viral DNA. In addition, the sera from 27 bushpigs and 126 domestic pigs were analysed with an ELISA test for the detection of antibodies against salivary antigens from Ornithodoros ticks. No circulation of ASFV or anti-ASFV antibodies nor anti-tick antibodies were detected in bushpigs. However, seven of the domestic pig sera (5.6% of the total sample population) were antibody positive for O. moubata antigens. The probability of freedom from ASFV in the bushpig population using Bayesian statistical methods ranged between 73% and 84%. The probabilities of absence of anti-tick antibodies in domestic and wild pigs were estimated at 63% and 71%, respectively. These preliminary results suggest that bushpigs are unlikely to play a significant role in the maintenance and transmission of ASFV in Madagascar. Nevertheless, further ASFV surveys are needed on that species to confirm this assumption. In addition, the presence of antibodies against O. moubata in domestic pigs suggests that soft ticks may be able to maintain ASFV within a domestic pig cycle in areas of Madagascar where they remain present.

Comparative proteomic analysis of Fasciola hepatica juveniles and Schistosoma bovis schistosomula.

Protein interactions between host and parasites can influence the infection success and severity. The aim of this investigation was to identify the proteins from two trematodes potentially localized at the host-parasite interface. We performed the proteomic profiles from in vivo obtained immature lung stage Schistosoma bovis schistosomula and in vitro excysted juveniles from Fasciola hepatica, parasites of ruminants and man usually giving rise to chronic infections. Proteomes from those parasites were obtained after digestion with trypsin and the peptides generated were identified by mass spectrometry, both before and after parasites' treatment with 70% methanol. The comparison of the two proteome sets from each parasite and between them, the analysis of their relative abundance and of their potential exposure to the host from living parasites, together with the specific immunolocalization of two of the identified molecules, show that this approach could assist in the identification of parasite exposed proteins and in the definition of molecules common for the two parasites with potential interaction with the host. Further characterization of these molecules could guide to define new common anti-parasitic targets and potential vaccine candidates.

The seroprevalence of human infection with Rickettsia slovaca, in an area of northern Spain.

An epidemiological survey was undertaken to explore human exposure to Rickettsia slovaca in two provinces of northern Spain. When IFAT were used to test 200 members of the general population for antibodies to rickettsiae of the spotted-fever group, six (3.3%) were found positive, presumably, since Dermacentor is one of the most common genera of human-biting tick in the study area, for antibodies to R. slovaca. Thirty-one (16.9%) of an additional 183 subjects who presented shortly after being bitten by ticks were also found seropositive. The difference in seroprevalence between the general and the tick-bitten populations was significant. Subject gender had no influence on seroprevalence in either population, although, in the tick-bitten group, age and occupation did have a significant influence on the prevalence recorded. Immunoblotting was used to confirm the presence of antibodies in the five subjects, all from the tick-bitten group, found to have acute infections. Three D. marginatus ticks obtained from three of these acute cases were found PCR-positive for R. slovaca DNA.

Analysis of the specificity of the salivary antigens of Ornithodoros erraticus for the purpose of serological detection of swine farms harbouring the parasite.

In Spain, considerable efforts are currently being devoted to the eradication of Ornithodoros erraticus from the swine farms harbouring this parasite, the European vector of African swine fever (ASF). However, to do so, a preliminary requirement is to determine on which farms it is present. Of all possible methods for discovering this, the only one feasible for large scale application is the serological detection of swine bearing anti-O. erraticus antibodies. To apply serology it was necessary to check the specificity of extracts from the salivary glands (SGE) from O. erraticus. For this, indirect ELISA, competitive ELISA and Western blot were used to assay the SGE from O. erraticus and their corresponding antisera against the SGE and respective antisera from 4 ixodidae, one mange mite, one louse and a mosquito. The results obtained show that only the anti-ixodidae sera are able to react against the SGE from O. erraticus. The cause of this reaction are the somatic antigens present in the SGE of the argasid but not its soluble (secretory) antigens. It is proposed that the anti-cement antibodies present in the anti-ixodidae sera are those that react with the somatic antigens of O. erraticus.

Canine filariasis in Salamanca (northwest Spain).

A survey of dogs in four areas of Salamanca province (northwest Spain) was carried out to determine the prevalence of filariids in the canine population. Blood samples from 293 dogs were tested for the presence of microfilariae, using a modification of the Knott technique. For correct identification of filariid species, blood smears associated with the 43 positive Knott tests were histochemically stained for demonstration of the somatic distribution of acid phosphatase activity. The survey revealed the presence in the northwest of Spain of Dirofilaria immitis, D. repens and Dipetalonema reconditum, with an overall prevalence in Salamanca of 12.3%, 0.3% and 2.1% respectively. The greater prevalence of canine heartworm was found in the irrigated area, where 33.3% of the dogs harboured microfilariae. The relationship between the prevalence of D. immitis and mosquito populations, and the influence of host sex and age in this infection, are analysed.