Compositional Attributes of Blue Lupin (Lupinus angustifolius) Seeds for Selection of High-Protein Cultivars.

Lupin is a promising protein source with a high protein concentration. Breeding efforts have resulted in the development of varieties low in quinolizidine alkaloids. The objective of this work was to evaluate 22 different blue lupin genotypes for a high protein concentration and low content of antinutritional alkaloids. These genotypes were grown under uniform controlled environmental and soil conditions, and the harvested seeds were evaluated for their composition. The low phosphorus content confirmed that the phytic acid presence was low in lupin, especially compared to other legumes. Furthermore, some of the varieties had less than 200 ppm alkaloids. Lupin proteins were rich in leucine and lysine, with the lowest amino acid concentration being methionine. There were significant differences in the protein concentration and recovery. This work demonstrated that an approach for selection of genotypes should be based on not only agronomic yields but also nutritional phenotypes, driving better decision making on future varietal selection.

Preparation and characterization of self-standing biofilms from compatible pectin/starch blends: Effect of pectin structure.

Pectin structure-miscibility-functionality relationships in starch films remain unknown. In this study, five citrus pectins (CPs) with 17 to 63 % of degree of methyl esterification (DM) and sugar beet pectin (SBP, rich in acetyl moieties and rhamnogalacturonan-I domains) were investigated for composition and structure and, further, blended with pea starch (3:1 starch-pectin weight ratio) to fabricate self-standing films. The incorporation of pectin resulted in a two- to three-fold increase in tensile strength and Young's modulus (up to 52.2 and 1837 MPa, respectively, using CP with low DM) without compromising elongation at break. Starch-SBP films presented the lowest strength among pectin films. Lower film moisture and water vapor permeability were attained with CP of high DM, or with SBP, whereas surface wettability was explained by counteracting factors affecting film compositional heterogeneity. Films made with high methoxyl CP, or with SBP, showed lower overall H-bonding (FTIR) and starch crystallinity (XRD). A DM above 57 % negatively affected the mixing and interfacial adhesion of pectin with starch, as shown by Attenuated Total Reflection-FTIR imaging. Pectins with the lowest purity, presumably with the greatest content in xyloglucan, as suggested by HPAEC, presented ~20 % higher elongation at break than the other films.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Electroconvulsive therapy use for refractory status epilepticus in an implantable vagus nerve stimulation patient: A case report.

Introduction: Status epilepticus (SE) has a mortality rate of 20 to 50%, with acute symptomatic SE having a higher risk compared to chronic SE. Electroconvulsive therapy (ECT) has been utilized for the treatment of refractory SE with a success rate estimate of 57.9%. There are no known reported cases of concomitant use of vagus nerve stimulation (VNS) and ECT for the treatment of super refractory SE (SRSE) available in the literature. Case description: We present a 44-year-old female with a history of developmental delay, epilepsy, an implantable VNS for 6 years, and traumatic brain injury with subsequent hygroma who presented with progressive aphasia, declining mental status, and daily generalized seizures lasting up to 20 min. Seizures had increased from her baseline of one seizure per day controlled with topiramate 200 mg three times daily and lamotrigine 400 mg twice daily. She was diagnosed with SRSE after being intubated and placed on eight anti-epileptic drugs (AEDs) that failed to abort SE. ECT was attempted to terminate SE. Due to a prior right craniotomy with subsequent right hygroma, eight treatments of ECT were performed over three sessions using a right anterior, left temporal (RALT) and subsequently a bitemporal electrode placement. The VNS remained active throughout treatment. Various ECT dosing parameters were attempted, varying pulse width and frequency. Although ECT induced mild transient encephalographic (EEG) changes following ECT stimulations, it was unable to terminate SE. Discussion: This case describes various treatment strategies, constraints, and device limitations when using ECT for the treatment of SE. With wide variability in efficacy rates of ECT in the treatment of SE in the literature, successful and unsuccessful cases offer information on optimizing ECT total charge dose and parameters that yielded success. This case demonstrates an instance of ECT inefficacy in the treatment of SRSE. Here, we discuss the rationale behind the various ECT settings that were selected, and constraints arising from the antiepileptic burden, VNS, and intrinsic limitations of the ECT device itself.

Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

Changes to fine structure, size and mechanical modulus of phytoglycogen nanoparticles subjected to high-shear extrusion.

This study aims to enhance the understanding of the structure of maize phytoglycogen nanoparticles, and the effect of shear scission on their architecture, radius, stiffness, and deformability. Compared to amylopectin, phytoglycogen had a lower A:B chain ratio, a lower number of chains per B chain, and a much higher number of Afingerprint chains. Phytoglycogen (Mw = 28.0 × 106 g/mol) was subjected to high-shear extrusion with varying Specific Mechanical Energies (SMEs) using different screw speeds, showing a maximum stable molecular weight Mw of ∼9.31 × 106 g/mol and a particle radius R reduction of 36 %, with a corresponding 20 % increase in the average mass density. Atomic force microscopy force spectroscopy revealed that nanoparticles extruded at the lowest SME (122 Wh/kg) exhibited a 20 % increase in Young's modulus. Higher SME values (up to 488 Wh/kg) resulted in an overall decrease in stiffness without further significant reductions in radius.

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model.

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

Pregelatinized Drum-Dried Wheat Starch of Different Swelling Behavior as Clean Label Oil Replacer in Oil-in-Water Emulsions.

Drum-drying results in pregelatinized starch with relatively low starch fragmentation and a great ability to absorb water and swell at room temperature. However, the effect of the degree of cold particle swelling and the thickening potential of drum-dried starch on its suitability as oil replacer in low-fat oil-in-water emulsions has received little attention. In this work, the potential of three pregelatinized drum-dried starches with almost identical molecular structure (as measured by size exclusion chromatography) and Water Binding Capacity (WBC), but different swelling behavior, was investigated to replace up to 60% oil in a mayonnaise-like emulsion system. The microstructure, stability, and rheology of the oil-in-water emulsions were noticeably affected by the substitution of oil with a pregelatinized drum-dried starch paste. Specifically, reduced-fat emulsions presented smaller droplet-size, a higher consistency index and increased emulsion stability, especially against freeze-thaw cycles, compared to the control full-fat mayonnaise. Importantly, the differences in cold swelling behavior (rather than simply assessing WBC) greatly influenced the consistency index and stability of low-fat emulsions, and results showed that drum-dried starch particles with high swelling potential perform better as oil replacers.

Case Report: Combined Liver-Kidney Transplantation to Correct a Mutation in Complement Factor B in an Atypical Hemolytic Uremic Syndrome Patient.

Pathogenic gain-of-function variants in complement Factor B were identified as causative of atypical Hemolytic Uremic syndrome (aHUS) in 2007. These mutations generate a reduction on the plasma levels of complement C3. A four-month-old boy was diagnosed with hypocomplementemic aHUS in May 2000, and he suffered seven recurrences during the following three years. He developed a severe hypertension which required 6 anti-hypertensive drugs and presented acrocyanosis and several confusional episodes. Plasma infusion or exchange, and immunosuppressive treatments did not improve the clinical evolution, and the patient developed end-stage renal disease at the age of 3 years. Hypertension and vascular symptoms persisted while he was on peritoneal dialysis or hemodialysis, as well as after bilateral nephrectomy. C3 levels remained low, while C4 levels were normal. In 2005, a heterozygous gain-of-function mutation in Factor B (K323E) was found. A combined liver and kidney transplantation (CLKT) was performed in March 2009, since there was not any therapy for complement inhibition in these patients. Kidney and liver functions normalized in the first two weeks, and the C3/C4 ratio immediately after transplantation, indicating that the C3 activation has been corrected. After remaining stable for 4 years, the patient suffered a B-cell non-Hodgkin lymphoma that was cured by chemotherapy and reduction of immunosuppressive drugs. Signs of liver rejection with cholangitis were observed a few months later, and a second liver graft was done 11 years after the CLKT. One year later, the patient maintains normal kidney and liver functions, also C3 and C4 levels are within the normal range. The 12-year follow-up of the patient reveals that, in spite of severe complications, CLKT was an acceptable therapeutic option for this aHUS patient.

Co-extruded wheat/okra composite blends result in soft, cohesive and resilient crumbs rich in health-promoting compounds.

This work investigates the partial solubilization of cell wall polysaccharides in okra flours and the changes in the profile of free and bound phenolics through twin-screw extrusion. The comparison between extruded wheat flour-native okra flour (EWF-OF) and extruded wheat flour-extruded okra flour (EWF-EOF) composite blends revealed that extrusion led to an increase of soluble dietary fiber from 7.76 to 10.02 g/100 g. Extrusion of okra also resulted in a significant increase of free and bound phenolic acids, the latter consisting mostly of ferulic acid, as well as the thermal degradation of free epigallocatechin, and the binding of a small portion of quercetin-3-O-glucoside likely to a carbohydrate fraction. Bread crumbs from EWF-EOF (at 15% replacement level) exhibited a significantly lower hardness and higher elasticity, cohesiveness and resilience (from 28.28 N, 0.94, 0.49 and 0.17 to 7.54 N, 0.99, 0.70 and 0.35, respectively), which closely resembled the textural attributes of wheat bread.

Distar Renal Tubular Acidosis (dRTA): Epidemiological, diagnostics, clinical follow-up and therapeutical issues. Nephrologists cohort survey outcome.

BACKGROUND AND OBJECTIVES: dRTA is a genetic or acquired rare disease, characterized by an unability to excrete hydrogens (H+) into urine, hypobicarbonatemia, hyperchloremia, and frequently hypercalciuria and hypokalaemia. Genetic forms are usually diagnosed during the first months of life and its treatment is based on providing alkali supplements in order to prevent long term clinical consequences, particularly chronic kidney disease (described in some cohorts up to 82% of dRTA patients) and the associated bone disease. A 10 queries multi choice closed response survey was designed to know more about epidemiological, diagnostics, clinical management and therapeutical issues of this disease among Spanish nephrologists. METHODS AND MATERIALS: This survey was delivered to the attendees to a scientific meeting on dRTA at the Spanish Nephrology Society congress in 2019. Surveys were collected at the end of this dRTA event. Results were analyzed by using a parametric statistical test, obtaining the percentage of each response for the 10 questions. RESULTS: Among the survey responders, 44.4% and 37.7% did not visit any dRTA patient during the 1st and 3rd last year respectively. When having a suspicious diagnose, confirming genetic diagnostic test is only performed on the 13.3% of cases and pedigree studies only on 11.1%. Only a 26.6% confirms that metabolic control is excellent, good or very good. 69% of the responders believe that treatment compliance is not bad, bad or very bad. CONCLUSIONS: This survey enhances the fact that dRTA is not a well known entity, satisfaction with metabolic control is poor and compliance is low. All these factors can lead to a higher severity of renal and bone diseases associated to dRTA.

The effect of extruded breadfruit flour on structural and physicochemical properties of beef emulsion modeling systems.

The objective was to determine structural and physicochemical properties of beef emulsion modeling systems prepared with native breadfruit flour and four different extruded breadfruit flours. Extrusion conditions for the flours were summarized as two different specific mechanical energies (74 or 145 kJ/kg) and four unique melt temperatures (83 °C, 100 °C, 105 °C, or 126 °C). Meat emulsions formulated at 3% replacement of beef with native or extruded breadfruit flours were compared with control (no additional flour) formulations. Replacement of beef with breadfruit flour (either native or extruded) did not significantly change cooking loss or instrumental redness values of cooked meat emulsions. Interestingly, replacement of beef with the fully gelatinized extruded breadfruit flours altered viscosity during heating as indicated by lower values for storage modulus (44.75% to 62.53% decrease compared with control) and lower values for loss modulus (25.90% to 52.54% decrease compared with control). This resulted in meat emulsions with a significant reduction in textural hardness (28.78% to 37.62% decrease compared with control).

Distar Renal Tubular Acidosis (dRTA): Epidemiological, diagnostics, clinical follow-up and therapeutical issues. Nephrologists cohort survey outcome. / Acidosis tubular renal distal (ATRd): aspectos epidemiológicos, diagnósticos, de seguimiento clínico y terapéuticos. Resultados de una encuesta a un colectivo de nefrólogos.

BACKGROUND AND OBJECTIVES: dRTA is a genetic or acquired rare disease, characterized by an unability to excrete hydrogens (H+) into urine, hypobicarbonatemia, hyperchloremia, and frequently hypercalciuria and hypokalaemia. Genetic forms are usually diagnosed during the first months of life and its treatment is based on providing alkali supplements in order to prevent long term clinical consequences, particularly chronic kidney disease (described in some cohorts up to 82% of dRTA patients) and the associated bone disease. A 10 queries multi choice closed response survey was designed to know more about epidemiological, diagnostics, clinical management and therapeutical issues of this disease among Spanish nephrologists. MATERIALS AND METHODS: This survey was delivered to the attendees to a scientific meeting on dRTA at the Spanish Nephrology Society congress in 2019. Surveys were collected at the end of this dRTA event. Results were analyzed by using a parametric statistical test, obtaining the percentage of each response for the 10 questions. RESULTS: Among the survey responders, 44.4% and 37.7% did not visit any dRTA patient during the 1st and 3rd last year respectively. When having a suspicious diagnose, confirming genetic diagnostic test is only performed on the 13.3% of cases and pedigree studies only on 11.1%. Only a 26.6% confirms that metabolic control is excellent, good or very good, and 69% of the responders believe that treatment compliance is not bad, bad or very bad. CONCLUSIONS: This survey enhances the fact that dRTA is not a well known entity, satisfaction with metabolic control is poor and compliance is low. All these factors can lead to a higher severity of renal and bone diseases associated to dRTA.

Modification of Physicochemical Properties of Breadfruit Flour Using Different Twin-Screw Extrusion Conditions and Its Application in Soy Protein Gels.

The objective was to modify functional properties of breadfruit flours using twin-screw extrusion and test the physicochemical properties of the extruded flours. Extruded breadfruit flours were produced with twin-screw extrusion using different last barrel temperature (80 °C or 120 °C) and feed moisture content (17% or 30%). These conditions resulted in four extruded flours with different mechanical (specific mechanical energy, SME) and thermal (melt temperature) energies. At temperatures below the gelatinization of the native starch (<70 °C), swelling power was increased in all extruded treatments. Solubility was dramatically increased in high-SME extruded flours at all tested temperatures. Water holding capacity was dramatically increased in the low-SME extruded flours. A two-fold higher cold peak viscosity was obtained for low SME-high temperature extruded flour compared with the other extruded flours. Low SME-low temperature extruded flour still exhibited a hot peak viscosity, which occurred earlier than in native flour. Setback was decreased in all extruded flours, especially in high-SME treatments. The incorporation of extruded flours into soy protein gels did not affect cooking loss, while hardness and springiness decreased with the addition of extruded flours. Overall, extrusion of breadfruit flour altered functional flour properties, including water holding capacity and pasting properties, and modified the texture of soy protein gels.

On the role of the internal chain length distribution of amylopectins during retrogradation: Double helix lateral aggregation and slow digestibility.

A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and Bfp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and 13C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6-12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and Bfp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.

High Temperature Rotational Rheology of the Seed Flour to Predict the Texture of Canned Red Kidney Beans (Phaseolus vulgaris).

The pasting profile of starchy tissues is conventionally measured by recording the apparent viscosity (η) in heating/cooling cycles. However, conventional rheometers show critical limitations when the starch is embedded in compact protein-rich cotyledon matrices, as occurs in pulses. In this work, the pasting profile of 13 red kidney beans (Phaseolus vulgaris) from the same cultivar but different growing locations was investigated using a heating/cooling cycle at higher temperature (130 °C) and pressurized conditions, using both water and brine as cooking solvents. It was hypothesized that the continuous measure of η at these conditions of flours from the dry seed would correlate with the texture, as determined by the mini-Kramer cell, of the beans after the entire process of soaking and canning. Furthermore, mechanistic answers were obtained by investigating their composition (starch, protein, and ash content) and physical properties (water holding capacity, seed ratio and weight). Interestingly, as opposed to the pasting profile at 95 °C, pasting indicators at 130 °C, including trough and final viscosity, strongly correlated with starch and protein content, seed coat ratio and, remarkably, with the firmness of the beans after canning when brine was incorporated. These results clearly show that small beans with a high protein content would bring about a more compact matrix that restricts starch from swelling and results in canned beans with a hard texture, which can be predicted by a lower pasting profile of the whole bean flour.

Comparative tachyzoite proteome analyses among six Neospora caninum isolates with different virulence.

The biological variability among Neospora caninum isolates has been widely shown, however, the molecular basis that determines this diversity has not been thoroughly elucidated to date. The latest studies have focused on a limited number of isolates. Therefore, the goal of the present study was to compare the proteome of a larger number of N. caninum isolates with different origins and virulence. Label-free LC-MS/MS was used to investigate the tachyzoite proteomic differences among Nc-Bahia, Nc-Spain4H and Nc-Spain7, representing high virulence isolates and Nc-Ger6, Nc-Spain2H and Nc-Spain1H, representing low virulence isolates. Pairwise comparisons between all isolates and between high virulence and low virulence groups identified a subset of proteins with higher abundance in high virulence isolates. These proteins were involved in energy and redox metabolism, and DNA/RNA processing, which might determine the faster growth rates and parasite survival of the high virulence isolates. Highlighted proteins included a predicted member of the rhoptry kinase family ROP20 specific for N. caninum, Bradyzoite pseudokinase 1 and several dense granule proteins. DNA polymerase, which was more abundant in all high virulence isolates in all comparisons, might also be implicated in virulence. These results reveal insights into possible mechanisms involved in specific phenotypic traits and virulence in N. caninum, and the relevance of these candidate proteins for N. caninum virulence deserves further investigation.