Unusual carriage of virulence genes sasX/sesI/shsA by nosocomial Staphylococcus haemolyticus from Brazil.

Background: Staphylococcus haemolyticus is an emerging threat in the nosocomial environment but only some virulence factors are known. Materials & methods: The frequency of the sasX gene (or orthologues sesI/shsA), encoding an invasiveness-related surface-associated protein, in S. haemolyticus was detected in different hospitals in Rio de Janeiro. Results: 9.4% of strains were sasX/sesI/shsA-positive, some were in the context of the ΦSPß-like prophage and devoid of CRISPR systems, indicating potential transferability of their virulence genes. Gene sequencing evidenced that Brazilian S. haemolyticus harbored sesI, instead of the usual sasX, while S. epidermidis had sasX instead of sesI, suggesting horizontal acquisition. Conclusion: The contexts of Brazilian sasX/sesI/shsA favor transfer, which is alarming given the difficulty in treating infections caused by S. haemolyticus.

Digital Mixed Content Analysis for the Study of Digital Platform Social Data: An Illustration from the Analysis of COVID-19 Risk Perception in the Italian Twittersphere.

The explosion of platform social data as digital secondary data, collectable through sophisticated and automatized query systems or algorithms, makes it possible to accumulate huge amounts of dense and miscellaneous data. The challenge for social researchers becomes how to extract meaning and not only trends in a quantitative and in a qualitative manner. Through the application of a digital mixed content analysis design, we present the potentiality of a hybrid digitalized approach to social content applied to a very tricky question: the recognition of risk perception during the first phase of COVID-19 in the Italian Twittersphere. The contribution of our article to mixed methods research consists in the extension of the existing definitions of content analysis as a mixed approach by combining hermeneutic and automated procedures, and by creating a design model with vast application potential, especially when applied to the digital scenario.

Niche availability and habitat affinities of the red porgy Pagrus pagrus (Linnaeus, 1758): An important ecological player on the world's largest rhodolith beds.

The red porgy (Pagrus pagrus) is a carnivore bottom dweller sparid, inhabiting flat sandy bottoms, rhodolith and seagrass beds of the Mediterranean Sea, the Western Atlantic (from Florida to Argentina) and the Eastern Atlantic (from Britain to Gabon). Along its native range, the red porgy is highly targeted by commercial and artisanal fisheries. In the past 40 years, the population decline of the species has been widely reported. In many locations, such as the Brazilian coast, stocks have collapsed. The central portion of the Brazilian coast harbours the largest rhodolith beds in the world and the highest levels of nektonic and benthic biodiversity. Along the rhodolith megahabitat, P. pagrus density is disproportionately higher (by 480%) than that of conspicuous benthic fishes inhabiting the same environment. Despite the ecological and economic importance of such an important species along its native range, little is known regarding its habitat use, niche availability and population responses to global warming. Here we present habitat affinities based on data sampled using baited remote stereo-video systems, and modelled niche availability and global warming populational responses. Our findings reveal that the red porgy is a species highly associated with rhodolith beds along the central portion of the Brazilian coast. The presence of a disproportional density and biomass of the red porgy, compared to other marine fish species, indicates that the species plays a key ecological role as a carnivore, mesoconsumer and prey/predator tolerant species, maintaining essential ecological functions in the habitat. In a global warming scenario, the model predicted populational niche shifts poleward and a severe niche erosion at lower latitudes as expected. Conservation initiatives (implementation of Maine Protected Areas, trawling exclusion zones, mining exclusion zones, fisheries management policies) are urgent to secure future stocks of the red porgy and also preserve the fragile rhodolith beds they inhabit.

Limited human access is linked to higher effectiveness in a marine sanctuary.

Marine Protected Areas (MPAs) are a widely used tool for coral reefs conservation, but massive tourism activities inside MPAs worldwide can challenge their effectiveness. This study investigated the role of different levels of protection strictness (no-entry, low and high tourism-allowed zones) set for a marine sanctuary in shaping benthic cover and reef fish community structure in the richest and largest coral reef system of the Southwestern Atlantic. Reef fish community structure and benthic cover differed between protection levels. No-entry zones showed significant higher coral coverage and biomass of piscivores and herbivores than tourism-allowed zones. Highest differences were found between no-entry and high tourism intensity zones. Despite the fact that protection from fishing by itself can ensure conservation benefits, we show here that the establishment of no-entry zones improve MPAs effectiveness.

Effects of seasonal contaminant remobilization on the community trophic dynamics in a Brazilian tropical estuary.

In tropical estuaries, wet seasons are responsible for the downstream transport of allochthonous material from the upper basin and flooded plains. Although allochthonous matter is commonly associated to nutrient and detritus input, pollutants are also transported throughout the basin or suspended from the river bottom via strong streamflow remobilization and rainfall dynamics. We assessed community and population trophic niche-based patterns using organisms' stable isotopes signatures in the wet and the dry seasons to test if estuarine trophic diversity is affected by remobilization of metal-contaminated material from a mining dam collapse that occurred in the Doce river basin, Brazil. Trophic depletion was detected community-wide and in a key consumer group (bottom-dwelling fishes) at the end of the wet season in the impacted Doce river estuary (DRE). Conversely, higher trophic diversity values were recorded in a well-preserved estuary used as control site. Stable isotopes mixing models indicated in the DRE that G. genidens, a predator fish species, presented poor-quality diet based on pollutant-tolerant tiny organisms, a finding that strongly contrasts from diet described in other, little-impacted Brazilian estuaries. Although wet seasons are expected to increase trophic, functional and taxonomic diversity in tropical estuaries, in the DRE the rainfall-driven dynamics poses a threat to the community due to the presence of ore tailings.

Resting egg banks can facilitate recovery of zooplankton communities after short exposure to glyphosate.

We assessed the short-term viability and recovery of zooplankton communities after exposure to glyphosate (active ingredient-a.i.). We conducted a hatching experiment in two steps: Step 1-natural lake sediments containing resting egg banks were placed into individual trays and exposed to a solution medium of glyphosate at three different treatments (LD = Values below the detection limits, LD < 0.05, 0.44, and 0.89 mg a.i./L) for 14 days; and Step 2-we replaced the exposure solution of glyphosate with distilled freshwater, keeping them all trays under freshwater conditions for another 14 day. The results from Step 1 showed significant effects of glyphosate on the emergence patterns of resting eggs, with a reduction in hatching of rotifers, mainly at concentrations of 0.44 and 0.89 mg a.i./L. On the other hand, the results from Step 2 showed an increase in the emergence of viable eggs for rotifers after restoration of freshwater conditions in all treatments; there was no effect for total zooplankton and microcrustaceans. These findings suggest that (i) glyphosate may, effectively, impair zooplankton hatching from resting egg banks; (ii) the magnitude of the negative effects depends on the the zooplanktonic group considered; and (iii) the restoration of freshwater conditions may, in some way, allow the recovery of the zooplankton community from viable egg banks. Our results can be useful in predicting the influence of glyphosate on the distribution patterns of freshwater zooplankton, which can represent vital information for environmental managers.

Early evidences of niche shifts in estuarine fishes following one of the world's largest mining dam disasters.

The Fundão dam collapse occurred on November 2015 in Mariana city (Brazil), provoking a series of ecological impacts over the Doce river basin and its nearshore environment. However many impacts over fishery target fauna (fish and shrimp) are still unknown or underestimated due to the lack of baseline data in the region. In the present study we assessed the isotopic niches modeled from δ13C and δ15N signatures of six estuarine fish species before and after the impact to assign potential shifts at the population- and community-level. We showed isotopic niche has altered in all studied species irrespective of its trophic group and habitat use. Niche community metrics indicated a depletion of trophic diversity and basal resources of the whole community after the impact. Food web changes as we reported here can impair the energy transfer through the food chain and put at risk the sustainability of small fisheries that rely upon local resources.

New records of the snow bass Serranus chionaraia (Perciformes: Serranidae) confirm an established population in the Brazilian Province.

The snow bass Serranus chionaraia is a small-bodied reef fish presumed to be restricted to the Caribbean Province, with a single specimen captured south of the Amazon River mouth. Recent surveys with baited remote underwater stereo-video systems detected the species c. 1900 km southward. Meristic and morphometric characters of two specimens examined in this study confirmed the species identity, which greatly extends the species' range southward on the eastern coast of Brazil and indicates the presence of an established population of S. chionaraia in the Brazilian Province.

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk.

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.

Evidence of Illegitimate Recombination Between Two Pasteurellaceae Plasmids Resulting in a Novel Multi-Resistance Replicon, pM3362MDR, in Actinobacillus pleuropneumoniae.

Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1 , sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required.

Mixture of commercial herbicides based on 2,4-D and glyphosate mixture can suppress the emergence of zooplankton from sediments.

It is generally assumed that zooplankton can recolonize lakes that have been exposed to pesticides, via their dormant egg banks. Hitherto, few studies have evaluated the relative importance of dormant egg bank recruitment in the re-establishment of zooplankton communities in the presence of pesticide. This study investigated the effects of commercial products Bratt® (a.i. 2,4-D), Roundup® (a.i. glyphosate) and their mixture on the emergence (abundance and taxon richness) of dormant zooplankton egg banks from natural lake sediment. Sediment samples were collected from the surface sediment (<10 cm depth) in four lakes in Southeast São Paulo, Brazil. We performed a hatching experiment, in which natural lake sediments containing dormant eggs were exposed separately to Bratt® (applied concentrations ranging from 0.30 to 20 mg L-1), Roundup® (0.28-8.5 mg L-1), and combined mixtures of all concentrations, plus one control (non-exposure to formulated herbicides) for a period of 28 days. All tested concentrations of Bratt®, Roundup® and their mixture reduced the abundance and taxon richness of emerging zooplankton (except 2 mg L-1 of Bratt®). This effect was more pronounced in rotifers. In comparison, there were no negative effects on the emergence of microcrustaceans. These findings suggest that commercial products Bratt®, Roundup® and their mixture can suppress the emergence of rotifers, thereby influencing zooplankton recruitment potential in lakes impacted by the presence of these commercial herbicides. Our results stress the importance of the need for additional studies to assess the effects of pesticides on dormant egg banks.

Ichthyofauna of the inner shelf of Paraná, Brazil: checklist, geographic distribution, economic importance and conservation status / Ictiofauna da plataforma interna do Paraná, Brasil: checklist, distribuição geográfica, importância econômica e estado de conservação

Abstract Comprehensive species checklists are essential to effectively implementing preservation and recovery measures, and should assess conservation status, vulnerability degree and anthropogenic threats. This checklist was compiled from fish species recorded in surveys conducted over the last 30 years in the shallow inner continental shelf in the State of Paraná, Brazil. Information on biogeography, conservation status, economic importance and degree of species' vulnerability are provided. A total of 272 fish species were recorded, spanning 25 orders and 88 families. The most speciose family was Sciaenidae (25 species), followed by Carangidae (23), Engraulidae (10), Carcharhinidae (9), Epinephelidae (9) and Paralichthyidae (9). Most species occurring in Paraná's shallow inner continental shelf are restricted to the West Atlantic. In terms of economic importance, 93% (253) of the species recorded were of fishery, aquaculture or aquaria interest. A notable fraction (39%) of the recorded species was classified as having moderate to very high vulnerability to extinction. In particular, 28 species (10%) were listed as globally endangered, and 34 species (12%) were listed as endangered in the Brazilian territory.

Resumo Listas abrangentes de espécies são essenciais para a implementação efetiva de medidas de preservação e devem avaliar o estado de conservação e o grau de vulnerabilidade das comunidades ecológicas. Esta lista foi compilada a partir de espécies de peixes registradas em pesquisas realizadas nos últimos 30 anos na plataforma continental interna rasa do Estado do Paraná. Foram fornecidas informações sobre biogeografia, estado de conservação, importância econômica e grau de vulnerabilidade das espécies. Um total de 272 espécies de peixes foram registradas, abrangendo 25 ordens e 88 famílias. A família com maior número de espécies foi Sciaenidae (25 espécies), seguida de Carangidae (23), Engraulidae (10), Carcharhinidae (9), Epinephelidae (9) e Paralichthyidae (9). A maioria das espécies registrada na plataforma continental interna do Paraná está limitada ao Atlântico Oeste. Em termos de importância econômica, 93% (253) das espécies incluídas nesta lista são comercialmente viáveis, seja na pesca, aquicultura ou aquariofilia. Uma fração notável (39%) das espécies registradas foi classificada como de vulnerabilidade moderada a muito alta. Em particular, 28 espécies (10%) estão listadas como ameaçadas de extinção em escala global, e 34 espécies (12%) estão listadas como ameaçadas de extinção no território brasileiro.

CRISPR-Cas Systems Features and the Gene-Reservoir Role of Coagulase-Negative Staphylococci.

The claimed role of gene reservoir of coagulase-negative staphylococci (CoNS) could be contradicted by estimates that CRISPR/Cas systems are found in the genomes of 40-50% of bacteria, as these systems interfere with plasmid uptake in staphylococci. To further correlate this role with presence of CRISPR, we analyzed, by computational methods, 122 genomes from 15 species of CoNS. Only 15% of them harbored CRISPR/Cas systems, and this proportion was much lower for S. epidermidis and S. haemolyticus, the CoNS most frequently associated with opportunistic infections in humans. These systems are of type II or III, and at least two of them are located within SCCmec, a mobile genetic element of Staphylococcus bacterial species. An analysis of the spacers of these CRISPRs, which come from exogenous origin, allowed us to track the transference of the SCCmec, which was exchanged between different strains, species and hosts. Some of the spacers are derived from plasmids described in Staphylococcus species that are different from those in which the CRISPR are found, evidencing the attempt (and failure) of plasmid transference between them. Based on the polymorphisms of the cas1 gene in CRISPRs of types II and III, we developed a multiplex polymerase chain reaction (PCR) suitable to screen and type CRISPR systems in CoNS. The PCR was tested in 59 S. haemolyticus strains, of which only two contained a type III cas1. This gene was shown to be expressed in the exponential growth, stationary phase and during biofilm formation. The low abundance of CRISPRs in CoNS is in accordance with their role as gene reservoirs, but when present, their spacers sequence evidence and give an insight on the dynamics of horizontal genetic transfer among staphylococci.

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae.

Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.

Transfer of mupirocin resistance from Staphylococcus haemolyticus clinical strains to Staphylococcus aureus through conjugative and mobilizable plasmids.

Coagulase-negative staphylococci are thought to act as reservoirs of antibiotic resistance genes that can be transferred to Staphylococcus aureus, thus hindering the combat of this bacterium. In this work, we analyzed the presence of plasmids conferring resistance to the antibiotic mupirocin-widely used to treat and prevent S. aureus infections in hospital environments-in nosocomial S. haemolyticus strains. About 12% of the 75 strains tested were resistant to mupirocin, and this phenotype was correlated with the presence of plasmids. These plasmids were shown to be diverse, being either conjugative or mobilizable, and capable of transferring mupirocin resistance to S. aureus Our findings reinforce that S. haemolyticus, historically and mistakenly considered as a less important pathogen, is a reservoir of resistance genes which can be transferred to other bacteria, such as S. aureus, emphasizing the necessity of more effective strategies to detect and combat this emergent opportunistic pathogen.

Skill-building through Task-Oriented Motor Practice (STOMP) intervention for activities of daily living: study protocol for a randomized, single blinded clinical trial.

BACKGROUND: Progressive disability in activities of daily living (ADL) is inevitable for people with Alzheimer's disease and related dementias (ADRD). Attempts to slow or prevent ADL disability have been unsuccessful despite making progress in behavioral training methods. Missing from this research is an emphasis on how we maximize a patient's engagement during training and the rigorous examination of implementation protocols (dosing and training methods) which may advantage learning in people with ADRD. Our team addressed this gap with the development of the STOMP (Skill-building through Task-Oriented Motor Practice) intervention which creates methods for obtaining ADL goals that support "personhood" and tests high-intensity protocols that appear to advantage learning and sustained learning over time. Through this study, we aim to evaluate differential outcomes by dose levels as well as assess the moderating effects of attention to task during training. METHODS/DESIGN: Randomized-controlled trial with 32 participants with dementia assigned to either the original, intensive STOMP protocol (3 hours/day, 5 days/week for 2 weeks) or a less-intensive STOMP protocol (1 hour/day, 2 days/week for 2 weeks) delivered by an occupational therapy assistant in the home. ADL training is delivered using motor learning theory techniques of blocked practice, continuous verbal praise, errorless learning and intense dosing schedules. Inclusion criteria: English speaking, adults 50-80 years old that live with a legally-authorized representative that can provide consent, who can follow a one-step command, have three ADL goals they want to address and can participate in an intense therapy protocol. Exclusions include diagnoses of Creutzfeldt-Jakob Dementia, delirium or receptive/global aphasia. Recruitment will occur through direct mailing, physician referral and media/support group presentations. Blinded occupational therapists will complete baseline, post-intervention and 3-month follow-up assessments in the home. Repeated measures ANOVA and graphs will be used to interpret and display results. DISCUSSION: Through this protocol, we will examine differential outcomes by dose for the STOMP ADL intervention. Our results will inform dosing parameters for future intervention studies for people with ADRD. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02356055. ETHICAL APPROVAL: This study protocol was approved by the University of Oklahoma Health Sciences Center Institutional Review Board (#4648) and will be performed in accordance with the Declaration of Helsinki.

User-centered design and usability testing of an innovative health-related quality of life module.

OBJECTIVES: Various computerized health risk appraisals (HRAs) are available, but few of them assess health-related quality of life (HRQoL) in a goal-directed framework. This study describes the user-centered development and usability testing of an innovative HRQoL module that extends a validated HRA tool in primary care settings. METHODS: Systematic user-centered design, usability testing, and qualitative methods were used to develop the HRQoL module in primary care practices. Twenty two patients and 5 clinicians participated in two rounds of interactive technology think-out-loud sessions (TOLs) and semi-structured interviews (SSIs) to iteratively develop a four-step, computerized process that collects information on patient goals for meaningful life activities and current level of disability and presents a personalized and prioritized list of preventive recommendations linked to online resources. RESULTS: Analysis of TOLs and SSIs generated 5 categories and 11 sub-categories related to facilitators and barriers to usability and human-technology interaction. The categories included: Understanding the Purpose, Usability, Perceived Value, Literacy, and Participant Motivation. Some categories were inter-connected. The technology was continually and iteratively improved between sessions until saturation of positive feedback was achieved in 4 categories (addressing motivation will require more research). Usability of all screen units of the module was improved substantially. Clinician feedback emphasized the importance of the module's ability to translate the patient-centered HRQoL Report into actionable items for clinicians to facilitate shared decision-making. Complete integration of the HRQoL module into the existing HRA will require further development and testing. CONCLUSIONS: Systematic application of user-centered design and human factors principles in technology development and testing may significantly improve the usability and clinical value of health information systems. This more sophisticated approach helped us translate complex clinical concepts, goal-setting steps, and decision-support processes into an accepted and value-added technology.

Comparing Differences in ADL Outcomes for the STOMP Intervention for Dementia in the Natural Home Environment Versus a Clinic Environment.

BACKGROUND: Few studies have examined structured rehabilitation techniques for improving activities of daily living in people with mild-moderate dementia. We sought to examine the advantages to delivering the Skill-building through Task-Oriented Motor Practice (STOMP) intervention in the home environment (versus the clinic), hypothesizing that ADL improvement would be significantly better, time to meeting goals would be faster and fewer displays of behavior would be noted. METHODS: Compared results of two quasi-experimental studies of STOMP, one completed in the home, one completed previously in a clinic. Participants were English-speaking; community dwelling adults aged 50-90 diagnosed with mild-moderate dementia who could participate in an intensive rehabilitation program (5 days/week, 3 hours/day, for 2 weeks). Outcome measurements include examiner-observation of performance and proxy-report of performance and satisfaction with performance in patient-selected goals. RESULTS: No differences existed in the sociodemographic characteristics between the home and clinic groups where the groups were primarily white, married, had > high school education and had mild-moderate dementia. Results from the home group indicate that participants made significant improvement in ADL which was generally retained at the 90 day follow-up. These results were not significantly different than the clinic group. No significant advantages were noted for the home group in terms of time to meeting goals or exhibition of fewer behaviors. DISCUSSION: The STOMP intervention appeared to work equally as well in the home and in the clinic. Future studies should continue to examine the benefits of massed practice using high-dose regimens.

A BOX-SCAR fragment for the identification of Actinobacillus pleuropneumoniae.

Bacterial respiratory diseases are responsible for considerable mortality, morbidity and economic losses in the swine industry. Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is one of the most important disease agents, but its identification and surveillance can be impaired by the existence of many other related bacteria in normal swine microbiota. In this work, we have evaluated a BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) sequence characterised amplified region (SCAR) marker for the specific identification of A. pleuropneumoniae and its use in a multiplex PCR to detect additionally Haemophilus parasuis and Pasteurella multocida, two other major respiratory pathogens of pigs that are members of the family Pasteurellaceae. PCRs based on the BOX-SCAR fragment developed were rapid, sensitive and differentiated A. pleuropneumoniae from all swine-related members of the Pasteurellaceae family tested. Single and multiplex BOX-SCAR fragment-based PCRs can be used to identify A. pleuropneumoniae from other bacterial swine pathogens and will be useful in surveillance and epidemiological studies.