Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

<p>Abstract</p> <p>Background</p> <p>Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of <it>Babesia bovis</it>, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival.</p> <p>Results</p> <p>Four papain-like cysteine proteases were found to be encoded by the <it>B. bovis </it>genome using the MEROPS database. One of them, the ortholog of <it>Plasmodium falciparum </it>falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in <it>B. bovis </it>chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several <it>B. bovis </it>isolates from North and South America. Synteny studies demonstrated that the <it>bovipain-2 </it>gene has expanded in the genomes of two related piroplasmids, <it>Theileria parva </it>and <it>T. annulata</it>, into families of 6 and 7 clustered genes respectively. The <it>bovipain-2 g</it>ene is transcribed in <it>in vitro </it>cultured intra-erythrocyte forms of a virulent and an attenuated <it>B. bovis </it>strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of <it>in vitro </it>cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with <it>B. bigemina</it>-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six <it>B. bovis</it>-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages.</p> <p>Conclusions</p> <p>Overall, we present the characterization of bovipain-2 and demonstrate its <it>in vitro </it>and <it>in vivo </it>expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.</p

Validation and field evaluation of a competitive enzyme-linked immunosorbent assay for diagnosis of Babesia bovis infections in Argentina

Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.Fil: Dominguez, Mariana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Zabal, Osvaldo Alfredo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Mosqueda, Juan J.. Universidad Autonoma de Queretaro.; MéxicoFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentin

Evaluation of the humoral and mucosal immune response of a multiepitope vaccine against COVID-19 in pigs

IntroductionThis study evaluated the immune response to a multiepitope recombinant chimeric protein (CHIVAX) containing B- and T-cell epitopes of the SARS-CoV-2 spike’s receptor binding domain (RBD) in a translational porcine model for pre-clinical studies.MethodsWe generated a multiepitope recombinant protein engineered to include six coding conserved epitopes from the RBD domain of the SARS-CoV-2 S protein. Pigs were divided into groups and immunized with different doses of the protein, with serum samples collected over time to determine antibody responses by indirect ELISA and antibody titration. Peptide recognition was also analyzed by Western blotting. A surrogate neutralization assay with recombinant ACE2 and RBDs was performed. Intranasal doses of the immunogen were also prepared and tested on Vietnamese minipigs.ResultsWhen the immunogen was administered subcutaneously, it induced specific IgG antibodies in pigs, and higher doses correlated with higher antibody levels. Antibodies from immunized pigs recognized individual peptides in the multiepitope vaccine and inhibited RBD-ACE2 binding for five variants of concern (VOC). Comparative antigen delivery methods showed that both, subcutaneous and combined subcutaneous/intranasal approaches, induced specific IgG and IgA antibodies, with the subcutaneous approach having superior neutralizing activity. CHIVAX elicited systemic immunity, evidenced by specific IgG antibodies in the serum, and local mucosal immunity, indicated by IgA antibodies in saliva, nasal, and bronchoalveolar lavage secretions. Importantly, these antibodies demonstrated neutralizing activity against SARS-CoV-2 in vitro.DiscussionThe elicited antibodies recognized individual epitopes on the chimeric protein and demonstrated the capacity to block RBD-ACE2 binding of the ancestral SARS-CoV-2 strain and four VOCs. The findings provide proof of concept for using multiepitope recombinant antigens and a combined immunization protocol to induce a neutralizing immune response against SARS-CoV-2 in the pig translational model for preclinical studies

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p&lt;0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p&lt;0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Radioactive characterization of leachates and efflorescences in the neighboring areas of a phosphogypsum disposal site as a preliminary step before its restoration

Trabajo presentado al "EU NORM 2 Symposium" celebrado en Praga (República Checa) del 17 al 19 de junio de 2014.En programa: "Preliminary Steps in the Restoration of a Phosphogypsum Disposal Site: Radioactive Characterization of Leachates and Efflorescences Affecting the Surrounding Area".After the recent closing of some phosphoric acid plants located in the South-West of Spain, it has been decided to restore a big extension (more than sixty hectares) of saltmarshes where historically some million tonnes of phosphogypsum (PG), the main byproduct generated by these plants, have been disposed of. This PG is characterized for containing high activity concentrations of several radionuclides from the uranium series, mainly 226Ra, 210Pb and 210Po and to a lesser extent U-isotopes. The PG disposal area can be considered as a potential source or radionuclides to their nearby environment, through the waters which percolate from them and through the efflorescences formed in their surroundings. A previous detailed radioactive characterization of the mentioned waters and efflorescences has been considered essential for a proper planification of the restoration plan to be applied in the near future in the zone.Peer Reviewe

Radiological and chemical risks by waste scales generated in the titanium dioxide industry

Along the industrial process devoted to the production of titanium dioxide pigments by using ilmenite as main raw material, small residues amounts are generated, remaining clearly enriched in natural radionuclides and chemical pollutants. Between them, we can remark the scales enriched in both radium isotopes and lead, which are formed in the internal walls of pipes and some equipment. These scales are radiological anomalies that demand its mineralogical, elemental and radiometric characterization as a basis for a detailed radiological and toxicological assessment from the occupational and public point of view. In this work, several scales collected in a TiO pigment production plant in South of Spain have been mineralogically characterized by XRD, while information about their elemental composition and morphology have been obtained by applying the XRF and SEM techniques. In addition, radiometric determinations have been performed by gamma-ray and alpha-particle spectrometry. The performed study indicates that the radiological doses received due to the scales by the workers performing its conventional activities are clearly lower than 1 mSv/y. Special dosimetric and chemical controls could be needed for the workers in charge of the maintenance labours (which include the removal of the scales) if these workers belong to an external company devoted to perform maintenance operations in several NORM industries.This research was partially supported by the Spanish Government Department of Science and Technology (MINECO) through the project “Fluxes of Radionuclides Emitted by the Phosphogypsum Piles Located at Huelva; Assessment of the Dispersion, Radiological Risks and Remediation Proposals” (Ref. CTM 2015-68628-R)), and the project of the Regional Government of Andalusia called “Basic processes regulating the fractionations and enrichments of natural radionuclides under acid mine drainage conditions” (Ref.: UHU-1255876) and the project of the University of Cadiz called “New methodogy of radioisotope isolation with environmental interest for its measurement by alpha spectrometry” (Ref: PR2019-024). The authors acknowledges to the staff of CITIUS at the University of Seville their help in the application of the X-ray techniques

Tick-borne Diseases in Syntopic Populations of Fallow Deer (\u3ci\u3eDama dama\u3c/i\u3e) and Axis Deer (\u3ci\u3eAxis axis\u3c/i\u3e) in Northern Mexico

We harvested 21 fallow deer (Dama dama) and 17 axis deer (Axis axis) in northern Mexico. Two fallow deer were positive for Babesia bigemina and one for Babesia bovis. Amplicons had the expected 170 and 291 base pairs and were identical to B. bigemina (S45366) and B. bovis (M38218), respectively

Prevalence of fleas and gastrointestinal parasites in free-roaming cats in central Mexico.

The prevalence of fleas and gastrointestinal parasites in free-roaming and domestic cats in central Mexico was evaluated. Three hundred and fifty eight cats captured in the street or brought in by owners to the Animal Control Center Unit, a unit of State Government, from June 2010 to May 2011, were included in the study. All cats were examined for the presence of fleas and gastrointestinal worms. One-hundred and ninety (53%) cats were infested with at least one flea species. Single infestations were observed in 106 (30%) cats and mixed infestations in 84 (23%) cats. Four species of fleas were recovered: Ctenocephalides felis in 53% of the cats, C. canis in 18%, Echidnophaga gallinacea in 7% and Pulex irritans in 1%. One-hundred and sixty three (45%) cats were infected with one or more species of gastrointestinal parasites: 48 (13%) with nematodes, 145 (40%) with cestodes, and one animal presented Moniliformis moniliformis. Prevalences and mean intensity of infection were: Physaloptera praeputialis 7 and 18; T. cati 3 and 2; Ancylostoma tubaeforme 2.5 and 2; Toxascaris leonina 0.5 and 2; Dipylidium caninum 36 and 32; Taenia taeniformis 4 and 3 and Moniliformis moniliformis 0.3 and 106, respectively. There was significant association (P<0.01), between season and ectoparasites load, more fleas were obtained in the summer and autumn than in the winter and spring; however, no statistical difference was observed for endoparasites load (P>0.05). The correlation between the total number of ectoparasites and endoparasites was not significant (r = 0.089, P = 0.094)

Cultivo in vitro de Anaplasma marginale en líneas celulares endoteliales

Anaplasma marginale, intracellular bacteria causes bovine anaplasmosis, characterized by progressive anemia, fever, decrease in body weight and death occasionally. The use of A. marginale in vitro cell culture - derived has been suggested as an alternative for development new prevention tools and control strategies.Anaplasma marginale, bacteria intracelular obligada, es el agente causal de la anaplasmosis bovina, caracterizada por anemia progresiva, fiebre, pérdida de peso y ocasionalmente, la muerte. Como alternativa para el desarrollo de nuevas estrategias de prevención se plantea el uso de cultivos celulares para la obtención in vitro de A. marginale