The evolution of constitutively active humoral immune defenses in Drosophila populations under high parasite pressure.

Both constitutive and inducible immune mechanisms are employed by hosts for defense against infection. Constitutive immunity allows for a faster response, but it comes with an associated cost that is always present. This trade-off between speed and fitness costs leads to the theoretical prediction that constitutive immunity will be favored where parasite exposure is frequent. We selected populations of Drosophila melanogaster under high parasite pressure from the parasitoid wasp Leptopilina boulardi. With RNA sequencing, we found the evolution of resistance in these populations was associated with them developing constitutively active humoral immunity, mediated by the larval fat body. Furthermore, these evolved populations were also able to induce gene expression in response to infection to a greater level, which indicates an overall more activated humoral immune response to parasitization. The anti-parasitoid immune response also relies on the JAK/STAT signaling pathway being activated in muscles following infection, and this induced response was only seen in populations that had evolved under high parasite pressure. We found that the cytokine Upd3, which induces this JAK/STAT response, is being expressed by immature lamellocytes. Furthermore, these immune cells became constitutively present when populations evolved resistance, potentially explaining why they gained the ability to activate JAK/STAT signaling. Thus, under intense parasitism, populations evolved resistance by increasing both constitutive and induced immune defenses, and there is likely an interplay between these two forms of immunity.

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite.

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We found that increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance.

Trans-regulatory changes underpin the evolution of the Drosophila immune response.

When an animal is infected, the expression of a large suite of genes is changed, resulting in an immune response that can defend the host. Despite much evidence that the sequence of proteins in the immune system can evolve rapidly, the evolution of gene expression is comparatively poorly understood. We therefore investigated the transcriptional response to parasitoid wasp infection in Drosophila simulans and D. sechellia. Although these species are closely related, there has been a large scale divergence in the expression of immune-responsive genes in their two main immune tissues, the fat body and hemocytes. Many genes, including those encoding molecules that directly kill pathogens, have cis regulatory changes, frequently resulting in large differences in their expression in the two species. However, these changes in cis regulation overwhelmingly affected gene expression in immune-challenged and uninfected animals alike. Divergence in the response to infection was controlled in trans. We argue that altering trans-regulatory factors, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion.

Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

Dissection of Besnoitia besnoiti intermediate host life cycle stages: From morphology to gene expression.

Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.

CO adsorption on MgO thin-films: formation and interaction of surface charged defects.

Two-dimensional (2D) materials formed by thin-films of metal oxides that grow on metal supports are commonly used in heterogeneous catalysis and multilayer electronic devices. Despite extensive research on these systems, the effects of charged defects at supported oxides on surface processes are still not clear. In this work, we perform spin-polarized density-functional theory (DFT) calculations to investigate formation and interaction of charged magnesium and oxygen vacancies, and Al dopants on MgO(001)/Ag(001) surface. The results show a sizable interface compressive effect that decreases the metal work function as electrons are added on the MgO surface with a magnesium vacancy. This surface displays a larger formation energy in a water environment (O-rich condition) even with additional Al-doping. Under these conditions, we found that a polar molecule such as CO is more strongly adsorbed on the low-coordination oxygen sites due to a larger contribution of the channeled electronic transport with the silver interface regardless of the surface charge. Therefore, these findings elucidate how surface intrinsic vacancies can influence or contribute to charge transfer, which allows one to explore more specific reactions at different surface topologies for more efficient catalysts for CO2 conversion.

The steps of thermal treatment of Na-magadiite: a computational study using ab initio DFT calculations.

We investigated each of the successive transformations of this material using ab initio calculations based on DFT. Possible structures produced from three reaction steps of the thermal treatment were simulated. Thermodynamic analysis was performed to assess the energy stability of each reaction. The dehydration of the interlamellar region confirmed the selective loss of water molecules, with axial H2O being responsible for the first part of the mass loss experimentally observed in TG-DTA while the loss of equatorial H2O molecules is observed above 150 °C. The reactions of the proposed intermediates after dehydration indicated that the formation of a zeolite Si14O28 is thermodynamically unfavorable in relation to zeolite sodium silicate. Kinetic effects and new heat treatment protocols should be studied to improve the understanding of these materials. The final steps indicated that after the condensation of the layers, sodium silicate was formed together with quartz.

Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids.

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster's immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution.

Parallel and costly changes to cellular immunity underlie the evolution of parasitoid resistance in three Drosophila species.

A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species.

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

Modulation of cis- and trans- Golgi and the Rab9A-GTPase during infection by Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum.

Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24 h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways.

Tetranychus urticae mites do not mount an induced immune response against bacteria.

The genome of the spider mite Tetranychus urticae, a herbivore, is missing important elements of the canonical Drosophila immune pathways necessary to fight bacterial infections. However, it is not known whether spider mites can mount an immune response and survive bacterial infection. In other chelicerates, bacterial infection elicits a response mediated by immune effectors leading to the survival of infected organisms. In T. urticae, infection by either Escherichia coli or Bacillus megaterium did not elicit a response as assessed through genome-wide transcriptomic analysis. In line with this, spider mites died within days even upon injection with low doses of bacteria that are non-pathogenic to Drosophila Moreover, bacterial populations grew exponentially inside the infected spider mites. By contrast, Sancassania berlesei, a litter-dwelling mite, controlled bacterial proliferation and resisted infections with both Gram-negative and Gram-positive bacteria lethal to T. urticae This differential mortality between mite species was absent when mites were infected with heat-killed bacteria. Also, we found that spider mites harbour in their gut 1000-fold less bacteria than S. berlesei We show that T. urticae has lost the capacity to mount an induced immune response against bacteria, in contrast to other mites and chelicerates but similarly to the phloem feeding aphid Acyrthosiphon pisum Hence, our results reinforce the putative evolutionary link between ecological conditions regarding exposure to bacteria and the architecture of the immune response.

Antimicrobial resistance and genomic rep-PCR fingerprints of Pseudomonas aeruginosa strains from animals on the background of the global population structure.

BACKGROUND: Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for fatal nosocomial infections worldwide, and has emerged as a relevant animal pathogen. Treatment options are dramatically decreasing, due to antimicrobial resistance and the microorganism's large versatile genome. Antimicrobial resistance profiles, serotype frequency and genomic profile of unrelated P. aeruginosa isolates of veterinary origin (n = 73), including domesticated, farm, zoo and wild animals mainly from Portugal were studied. The genomic profile, determined by DiversiLab system (Rep-PCR-based technique), was compared with the P. aeruginosa global population structure to evaluate their relatedness. RESULTS: Around 40% of the isolates expressed serotypes O6 (20.5%) and O1 (17.8%). A total of 46.6% of isolates was susceptible to all antimicrobials tested. Isolates obtained from most animals were non-multidrug resistant (86.3%), whereas 11% were multidrug resistant, MDR (non-susceptible to at least one agent in ≥ three antimicrobial categories), and 2.7% extensively drug resistant, XDR (non-susceptible to at least one agent in all but two or fewer antimicrobial categories). Resistance percentages were as follows: amikacin (0.0%), aztreonam (41.1%), cefepime (9.6%), ceftazidime (2.7%), ciprofloxacin (15.1%), colistin (0.0%), gentamicin (12.3%), imipenem (1.4%), meropenem (1.4%), piperacillin + tazobactam (12.3%), ticarcillin (16.4%), ticarcillin + clavulanic acid (17.8%), and tobramycin (1.4%). Animal isolates form a population with a non-clonal epidemic structure indistinguishable from the global P. aeruginosa population structure, where no specific 'animal clonal lineage' was detected. CONCLUSIONS: Serotypes O6 and O1 were the most frequent. Serotype frequency and antimicrobial resistance patterns found in P. aeruginosa from animals were as expected for this species. This study confirms earlier results that P. aeruginosa has a non-clonal population structure, and shows that P. aeruginosa population from animals is homogeneously scattered and indistinguishable from the global population structure.

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response.

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.

Related strains of African swine fever virus with different virulence: genome comparison and analysis.

Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Thermally activated surface oxygen defects at the perimeter of Au/TiO2: a DFT+U study.

Density functional theory calculations were performed to examine the formation of oxygen atom vacancies on three model surfaces namely, clean anatase TiO2(001) and, Au3 and Au10 clusters supported on anatase TiO2(001). On the Au/TiO2 systems, three different types of lattice oxygen atoms can be identified: the Ti-O-Au bridge, the Ti-O-Ti bridge in the perimeter of the Au cluster and the Ti-O-Ti bridge away from the Au cluster, the oxygen atoms on the clean surface. The variation in ΔG° with temperature for surface O vacancy formation was calculated for these three situations using total-energy, vibrational structure and optimized geometries of the material surfaces and the O2 molecule. The calculations reveal that the O defect formation on the clean anatase TiO2(001) surface seems very difficult due to the large positive value of ΔG° (290 kJ mol(-1)) from 0 to 650 K. However, the presence of the Au cluster on the TiO2 surface changes the surface chemistry of the TiO2 significantly. We observed that the trend in ΔG° variation for the vacancy formation from the Ti-O-Au bridge is the same as on Au3/TiO2 and Au10/TiO2 systems, almost constant with large positive values of ΔG° around 250 and 350 kJ mol(-1), respectively. The ΔG° for the perimeter defect formation (Ti-O-Ti bridge in the perimeter of the Au cluster) is smaller for Aun/TiO2 systems than the clean TiO2 surface, however, the vacancy formation is possible only for the Au10/TiO2 system (close to 506 K). Finally, extended calculations for other oxygen atoms on the Au10/TiO2 model reveal that the trend in ΔG° variation is similar for all the interface or perimeter O atoms around the Au cluster with marginal differences in the numerical value of ΔG°. Since, the surface O atoms are activated only in the presence of a particular sized Au, we propose that a Au catalyzed Mars-van Krevelen mechanism could be a possible reaction mechanism for CO oxidation on Au/TiO2 catalysts at slightly elevated temperatures.

An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins.

Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02EU/µg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.

A review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by Besnoitia besnoiti (Franco and Borges, ).

Bovine besnoitiosis is caused by the largely unexplored apicomplexan parasite Besnoitia besnoiti. In cows, infection during pregnancy often results in abortion, and chronically infected bulls become infertile. Similar to other apicomplexans B. besnoiti has acquired a largely intracellular lifestyle, but its complete life cycle is still unknown, modes of transmission have not been entirely resolved and the definitive host has not been identified. Outbreaks of bovine besnoitiosis in cattle were described in the 1990s in Portugal and Spain, and later several cases were also detected in France. More cases have been reported recently in hitherto unaffected countries, including Italy, Germany, Switzerland, Hungary and Croatia. To date, there is still no effective pharmaceutical compound available for the treatment of besnoitiosis in cattle, and progress in the identification of novel targets for intervention through pharmacological or immunological means is hampered by the lack of molecular data on the genomic and transcriptomic level. In addition, the lack of an appropriate small animal laboratory model, and wide gaps in our knowledge on the host-parasite interplay during the life cycle of this parasite, renders vaccine and drug development a cost- and labour-intensive undertaking.