Artículos Científicos

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Activation of Rho and Rab GTPases dissociates Brucella abortus internalization from intracellular trafficking
(Blackwell Science, 2002-10-04) Chaves-Olarte, Esteban; Guzman-Verri, Caterina; Méresse, Stephane; Desjardins, Michel; Pizarro-Cerdá, Javier; Badilla, Jenny; Gorvel, Jean-Pierre; Moreno, Edgardo
Brucella abortus is an intracellular pathogen that relies on unconventional virulence factors to infect hosts. In non-professional phagocytes, Rho GTPasesactivation by the Escherichia coli cytotoxic necrotiz ing factor (CNF) promoted massive Brucella entrance by membrane ruffling, a mechanism that differs from the common mode of entrance used by this bacterium in non-treated cells. Cytotoxic necrotizing factor treatment, however, did not alter the intracellular route followed by the wild type or non-virulent defined mutants. In contrast, expression of a constitutively active Rab5Q79L GTPase did not alter cell-invasion by Brucella but hampered its ability to reach the endoplasmic reticulum. The CNF-induced Brucella superinfection did not reduce the ability of host cells to synthesize DNA and progress through the cell cycle. Furthermore, CNF-treatment increased the isolation of Brucella-containing compartments by a factor of 15. These results demonstrate that in nonprofessional phagocytic cells, Brucella manipulates two different sets of GTPases during its biogenesis, being internalization and intracellular trafficking two consecutive but independent processes. Besides, CNF-induced super-infection demonstrates that Brucella does not interfere with crucial cellular processes and has shown its potential as tool to characterize the intracellular compartments occupied by this bacterium.
Asociación entre variables: correlación no paramétrica
(Colegio de Médicos y Cirujanos, Costa Rica, 2010-03-09) Camacho Sandoval, Jorge
Se describe la forma de estimar el coeficiente de correlación de Spearman y las condiciones en que resulta apropiada su utilización. También se describe como realizar una prueba de hipótesis para determinar si el coeficiente estimado es significativamente distinto de cero.
Brucella abortus senses the intracellular environment through the BvrR/Bvrs two-component system, which allows B. abortus to adapt to its replicative nich
(American Society for Microbiology., 2018-01-16) Altamirano-Silva, Pamela; Meza-Torres, Jazmín; Castillo-Zeledón, Amanda; Ruiz-Villalobos, Nazareth; Zúñiga Pereira, Ana Mariel; Chacón-Díaz, Carlos; Moreno, Edgardo; Guzman-Verri, Caterina; Chaves-Olarte, Esteban
Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of Alphaproteobacteria
Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection
(PLOS ONE, 2007-07-18) BARQUERO-CALVO, ELIAS; Chaves-Olarte, Esteban; Weiss, David; Guzman-Verri, Caterina; Chacón-Díaz, Carlos; Rucavado, Alexandra; Moriyon, Ignacio; Moreno, Edgardo
Background. To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings. Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-a-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance. We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.
Brucella genetic variability in wildlife marine mammals populations relates to host preference and ocean distribution
(Oxford University Press on behalf of the Society for Molecular Biology and Evolution, 2017-07-20) Suárez-Esquivel, Marcela; Baker, Kate; Ruiz-Villalobos, Nazareth; Hernandez-Mora, Gabriela; BARQUERO-CALVO, ELIAS; González-Barrientos, Rocío; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Chacón-Díaz, Carlos; Cloeckaert, Axel; Chaves-Olarte, Esteban; Thomson, Nicholas; Moreno, Edgardo; Guzman-Verri, Caterina
Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellularintracellularbacteriaresponsibleforcausing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97–99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication.
Brucellosis in mammals of Costa Rica: an epidemiological survey
(PLOS ONE, 2017-08-09) Hernández-Mora, Gabriela; Bonilla-Montoya, Roberto; Barrantes-Granados, Osvaldo; Esquivel-Suárez, Andrea; Montero-Caballero, Danilo; Fallas-Monge, Zeanne; Palacios Alfaro, Jose David; Baldi, Mario; Campos, Elena; Chanto, Grettel; BARQUERO-CALVO, ELIAS; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzman-Verri, Caterina; Romero-Zúñiga, Juan José; Moreno, Edgardo
La brucelosis ha sido una enfermedad endémica del ganado y los seres humanos en Costa Rica desde principios del siglo XX. Sin embargo, no se ha reportado brucelosis en ovejas, cabras, cerdos, búfalos de agua, caballos y cetáceos en el país. Hemos realizado un estudio de brucelosis en estas especies de mamíferos hospedadores, de 1999 a 2016. Además, hemos documentado el número de casos notificados de brucelosis humana entre 2003 y 2016. La seroprevalencia de brucelosis en rebaños de cabras y ovejas fue de 0,98% y 0,7% respectivamente, sin aislamiento de Brucella . No se detectaron anticuerpos contra Brucella en cerdos domésticos o salvajes. Asimismo, la seroprevalencia de brucelosis en granjas de caballos y búfalos de agua se estimó en 6.5% y 21.7%, respectivamente, sin Brucellaaislamiento. Seis especies de cetáceos mostraron reacciones positivas contra Brucella antígenos, y B . ceti se aisló en el 70% (n = 29) de los delfines rayados ( Stenella coeruleoalba ). Se observó un aumento constante en el diagnóstico de casos de brucelosis humana. Teniendo en cuenta la prevalencia de brucelosis en los distintos mamíferos hospedadores de Costa Rica, se recomiendan diferentes medidas.
BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence
(American Society for Microbiology, 2007-07-30) Manterola, Lorea; Guzman-Verri, Caterina; Chaves-Olarte, Esteban; BARQUERO-CALVO, ELIAS; de Miguel, María-Jesús; Moriyon, Ignacio; Grillo, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo
The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps.
Coeficiente de concordancia para variables continuas
(Colegio de Médicos y Cirujanos, Costa Rica, 2010-03-09) Camacho Sandoval, Jorge
En las notas estadísticas anteriores, se hizo referencia a medidas de asociación entre dos variables. En el caso particular de dos variables continuas, se estudió el coeficiente de correlación de Pearson y su forma de cálculo. Una situación diferente surge cuando se desea medir la misma variable, en las mismas muestras o pacientes, con dos métodos, equipos o personas diferentes, para determinar si ambos métodos, equipos o personas producen resultados equivalentes. En ese caso lo que interesa es determinar si ambas mediciones son similares en magnitud, no si están asociadas, de hecho deben estarlo, ya que son dos mediciones de la misma característica en los mismos individuos o muestras. No obstante, si una de las mediciones tiene un error sistemático, por ejemplo si una de las mediciones tiene sistemáticamente cinco unidades menos que la otra medición, el coeficiente de correlación puede ser muy elevado aunque las diferencias en las mediciones sean importantes, es decir, las mediciones pueden no ser concordantes.
Combined electrokinetic manipulations of pathogenic bacterial samples in low-cost fabricated dielectrophoretic devices
(AIP Advances, 2019-11-13) Martinez-Brenes, Alejandro; Torres-Castro, Karina; Marín-Benavides, Richard; Acuña-Umaña, Katherine; Espinoza Araya, Christopher; Ramírez-Carranza, Raquel; González-Espinoza, Gabriela; Rojas-Campos, Norman; Guzman-Verri, Caterina; Sáenz-Arce, Giovanni; Lesser-Rojas, Leonardo
A low-cost fabrication method of microfluidic devices with micrometer-sized constrictions used for electrodeless dielectrophoresis (eDEP) is demonstrated here. A structure on a commercial printed circuit board (PCB) template of one-sided copper clad fiberglass-epoxy laminate was used as a molding master for polydimethylsiloxane (PDMS) soft lithography. This was achieved by printing a constriction-based microchannel pattern on glossy paper with a micrometer-scaled resolution laser printer and transferring it to the laminate’s Cu face, rendering a microstructure of ∼17 μm height and various widths across tips. The Cu master’s pattern was transferred to PDMS, and smooth constrictions were observed under the microscope. Following air plasma encapsulation, PDMS chips were loaded with an inactivated bacterial sample of fluorescently stained Brucella abortus vaccine strain S-19 and connected to an amplified voltage source to examine the sample’s response to electric field variations. After an AC/DC electric field was applied to the bacterial solution in the microfluidic device, the combined effect of electrokinetic + hydrodynamic mechanisms that interact near the dielectric microconstrictions and exert forces to the sample was observed and later confirmed by COMSOL simulations. Our fabrication method is an alternative to be used when there is no access to advanced microfabrication facilities and opens ways for target selection and preconcentration of intracellular pathogens as well as sample preparation for metagenomics.
Distribución del Pequeño Escarabajo de la Colmena, Aethina tumida, en abejas africanizadas (Apis mellifera) en diferentes zonas apícolas de Costa Rica
(Universidad Nacional, Costa Rica, 2020-07-01) Arguedas Mora, Martha; Soto González, Juan Francisco; Ramírez Montero, Marianyela; Calderón Fallas, Rafael A.
El primer reporte sobre la presencia del Pequeño Escarabajo de la Colmena (PEC), Aethina tumida, en Costa Rica, se realizó el 25 de agosto 2015, en La Cruz, Guanacaste; lo cual aumenta el riesgo de dispersión a otras zonas apícolas del país. Por tanto, para este estudio, se realizó un muestreo de mayo a octubre del 2019. Se analizó 305 colmenas, distribuidas en 61 apiarios, ubicados en San Ramón, Alajuela. Adicionalmente, se examinó muestras de escarabajos adultos provenientes de Ulloa, Heredia y de Potrerillos de Liberia, Guanacaste. En cada apiario se analizó, aleatoriamente, cinco colmenas. Se revisó la tapa, los marcos, las cajas y el piso, con el fin de identificar la presencia de huevos, larvas y/o adultos del escarabajo. También, para reconocer algún signo relacionado, como galerías en los panales, apariencia húmeda de las abejas, entre otros. Las muestras de escarabajos adultos se analizaron en el laboratorio de Patología Apícola del CINAT-UNA. Las 305 colmenas evaluadas, en San Ramón de Alajuela, resultaron negativas para el Pequeño Escarabajo. Por otra parte, se confirmó la presencia de A. tumida en una colonia establecida en una casa de habitación en Ulloa, Heredia; también, en un apiario ubicado en Potrerillos de Liberia, el cual se encuentra aproximadamente a 25 km del foco inicial en La Cruz, Guanacaste. Con base en lo expuesto, se puede indicar la presencia de A. tumida en colmenas de abejas africanizadas en diferentes zonas de Costa Rica, con una distribución limitada. Al considerar que el escarabajo adulto puede volar y desplazarse, junto a enjambres silvestres; además, la trashumancia de colmenas por parte del apicultor, se debe mantener una revisión periódica de los apiarios, dado que las condiciones actuales de ausencia, en la mayoría de colmenas analizadas en este estudio, pueden cambiar.
Enfermedad de Chagas aguda, en un adulto tratado con benznidazol (Rochagan®) en Costa Rica
(Colegio de Médicos y Cirujanos, Costa Rica, 2004-04-01) Montenegro Hidalgo, Víctor Manuel; Castro Ugalde, Jorge; Zeledón, Rodrigo
Se da a conocer un caso agudo de la enfermedad de Chagas en un adulto de 48 años con signo de Romaña, infectado al recibir un chorro de orina de una chinche (T. dimidiata), cuando trataba de destriparla con un palo de café. Es el primer adulto con enfermedad de Chagas aguda tratado en Costa Rica con benznidazol. Se citan los síntomas encontrados, así como los aspectos epidemiológicos relacionados con su presentación. Se enfatizan el tratamiento y la evolución del caso. Se hace un llamado de atención a los clínicos, ya que este caso no fue diagnosticado apropiadamente desde un inicio, lo que evidencia el desconocimiento que existe de la enfermedad de Chagas como motivo de consulta.
Epidemiology of bovine brucellosis in Costa Rica: Lessons learned from failures in the control of the disease
(PLOS ONE, 2017-08-10) Hernández-Mora, Gabriela; Ruiz-Villalobos, Nazareth; Bonilla-Montoya, Roberto; Romero-Zúñiga, Juan José; Jiménez-Arias, Julio; González-Barrientos, Rocio; BARQUERO-CALVO, ELIAS; Chacón-Díaz, Carlos; Rojas, Norman; Chaves-Olarte, Esteban; Guzman-Verri, Caterina; Moreno, Edgardo
Brucellosis, caused by Brucella abortus is a major disease of cattle and a zoonosis. In order to estimate the bovine brucellosis prevalence in Costa Rica (CR), a total 765 herds (13078 bovines) from six regions of CR were randomly sampled during 2012–2013. A non-random sample of 7907 herds (532199 bovines) of the six regions, arriving for diagnoses during 2014–2016 to the Costa Rican Animal Health Service was also studied. The prevalence estimated by Rose Bengal test (RBT) ranged from 10.5%-11.4%; alternatively, the prevalence estimated by testing the RBT positives in iELISA, ranged from 4.1%-6.0%, respectively. However, cattle in CR are not vaccinated with B. abortus S19 but with RB51 (vaccination coverage close to 11%), and under these conditions the RBT displays 99% specificity and 99% sensitivity. Therefore, the RBT herd depicted in the random analysis stands as a feasible assessment and then, the recommended value in case of planning an eradication program in CR. Studies of three decades reveled that bovine brucellosis prevalence has increased in CR. B. abortus was identified by biochemical and molecular studies as the etiological agent of bovine brucellosis. Multiple locus variable-number tandem repeat analysis-16 revealed four B. abortus clusters. Cluster one and three are intertwined with isolates from other countries, while clusters two and four have only representatives from CR. Cluster one is widely distributed in all regions of the country and may be the primary B. abortus source. The other clusters seem to be restricted to specific areas in CR. The implications of our findings, in relation to the control of the disease in CR, are critically discussed.
Human neutrophils are resistant to Clostridioides difficile toxin B
(Elsevier, 2022-03-21) Chaves-Cordero, Catalina; Quesada-Gómez, Carlos; Chaves-Olarte, Esteban; BARQUERO-CALVO, ELIAS
Objective: The main objective of this study was to evaluate the glucosyltransferase activity of C. difficile TcdB on the activity of human PMNs. Methods: To better understand the interaction between PMNs and TcdB, PMNs were treated with sublethal concentrations of TcdB. We evaluated: (i) the glucosylation of GTPases, (ii) the phagocytic and bactericidal activity, and (iii) PMNs activation (through quantification of TNF-a, IL-8, and expression of CD11b cell surface activation marker). Results: We found that TcdB did not glucosylate RhoA and Rac1 GTPases and did not affect the phagocytic or bactericidal capacity of PMNs. Moreover, TcdB did not increase the production of TNF-a, IL-8, or the expression of activation marker CD11b. The only significant effect of TcdB on PMNs was the partial inhibition of TNF-a and IL-8 production and the diminished expression of CD11b induced by E. coli-LPS. Conclusion: Our results show that human PMNs are resistant to TcdB GTPase glucosyltransferase activity against RhoA and Rac1.
In vivo proteolytic degradation of the Escherichia coli acyltransferase HlyC
(The American Society for Biochemistry and Molecular Biology, 2001-05-18) Guzman-Verri, Caterina; Chaves-Olarte, Esteban; García, Fernando; Arvidson, Staffan; Moreno, Edgardo
Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as “repeats in toxins.” Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.
Intracellular adaptation of Brucella abortus
(Universidad Nacional, Costa Rica, 2009-03) Lamontagne, Julie; Forest, Anik; Marazzo, Elena; François, Denis; Butler, Heather; Michaud, Jean-François; Boucher, Lyne; Pedro, Ida; Villeneuve, Annie; Sitnikov, Dmitri; Trudel, Sylvie; Nassif, Najib; Boudjelt, Djamila; Tomaki, Fadi; Chaves-Olarte, Esteban; Guzman-Verri, Caterina; Brunet, Sylvain; Côté-Martin, Alexandra; Hunter, Joanna; Moreno, Edgardo; Paramithiotis, Eustache
Macrophages were infected with virulent B. abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration, adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain’s adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections
Investigación, poblaciones y muestras
(Colegio de Médicos y Cirujanos, Costa Rica, 2010-03-09) Camacho Sandoval, Jorge
Usualmente, los investigadores en ciencias de la salud están interesados en una población, formada por un conjunto de individuos, con uno o más atributos comunes. Por ejemplo, la población de pacientes de Diabetes Mellitus no insulinodependientes, de ambos sexos, con edades comprendidas entre 18 y 60 años, residentes en el área de atracción del Hospital México.
Isolation and identification of Arcobacter species from Costa Rican poultry production and retail sources
(Journal of food protection, 2017-04-03) Zumbado, Leana; Barboza, Karol; Angulo, Irina; Redondo-Solano, Mauricio; Castro, Eduardo; Arias, María Laura
Arcobacter is a gram-negative rod recognized as a potential food- and waterborne pathogen; nevertheless, little is known about the effects of this pathogen on human and animal health. Although Arcobacter species are commonly found in nature, poultry is suspected to be the main vehicle for the transmission of this pathogen. The aims of this work were to determine the prevalence of Arcobacter spp. in broilers produced in Costa Rica for human consumption and to analyze the pathogenic capacity of the isolates through the detection of virulence genes. One hundred fifty-two samples of cecal content (87 farms), 104 samples of carcass rinse after chiller (six processing plants), and 96 carcass rinses from as many retail stores were analyzed. The suspicious isolates were identified using genus-specific PCR, and species-level identification was achieved with a multiplex PCR. Virulence genes were identified using the protocol described by L. Douidah, L. de Zutter, J. Baré, P. De Vos, P. Vandamme, O. Vandenberg, A.-M. Van den Abeele, and K. Houf (J. Clin. Microbiol. 50:735–741, 2012), which includes nine different virulence genes. The overall isolation frequency of Arcobacter was 6.5% (n = 23). Eight (34.8%) of the isolates came from cecal content, 2 (8.7%) were isolated from samples taken after chiller, and 13 (56.5%) were from retail stores. The species isolated included A. thereius (30.4%), A. butzleri (21.7%), A. skirrowii (4.3%), and A. cibarius (4.3%). The remaining samples were classified as Arcobacter sp. Gene tlyA was the most prevalent virulence gene, present in 9 of 23 samples analyzed; genes hecA and pldA were present in one only strain each. A strain of A. butzleri isolated from a retail store presented the highest number of virulence genes (five), and 11 samples did not present any of the genes analyzed. The results obtained suggest that the presence of virulent Arcobacter isolates in the poultry production chain from Costa Rica could be a risk for individuals who consume the contaminated product.
Isolierung von Coronaviren in der Zellkultur aus Nasentupferproben atemwegskranker Kalber in der Bundesrepublik Deutschland
(Paul Parey Scientific Publishers, 1989) Jiménez, Carlos; Herbst, W; Müller, J.M; SCHLIESSER, TH
Isolation in tissue culture of coronavirus from respiratory diseased calves in the FRG Coronavirus was isolated in HRT-IS tissue culture from nasal swabs of 4 from 15 calves showing respiratory symptoms. The strains showed cytopathic effects and could be identified as coronaviruses of bovine origin by their mouse erythrocyte agglutinating activity as well as serological specific hemagglutination inhibition and electron microscopical methods. This is the first report on the isolation of coronaviruses in tissue culture from the respiratory tract of calves in the FRG.
Long-term in vivo performance of low-temperature 3D-printed bioceramics in an equine model
(American Chemical Society, 2020-01-31) Vindas Bolaños, Rafael; Castilho, Miguel; de Grauw, Janny; Cokelaere, Stefan M.; Plomp, Saskia; Groll, Jürgen; van Weeren, René; Gbureck, Uwe; Malda, Jos
Bone has great self-healing capacity, but above a certain critical size, bone defects will not heal spontaneously, requiring intervention to achieve full healing. Among the synthetic calcium phosphate (CaP) bone replacement materials, brushite (CaHPO4·2H2O)-based materials are of particular interest because of their degree of solubility and the related high potential to promote bone regeneration after dissolution. They can be produced tailor-made using modern three-dimensional (3D) printing technology. Although this type of implant has been widely tested in vitro, there are only limited in vivo data and less so in a relevant large animal model. In this study, material properties of a 3D-printed brushite-based scaffold are characterized, after which the material is tested by in vivo orthotopic implantation in the equine tuber coxae for 6 months. The implantation procedure was easy to perform and was well tolerated by the animals, which showed no detectable signs of discomfort. In vitro tests showed that compressive strength along the vertical axis of densely printed material was around 13 MPa, which was reduced to approximately 8 MPa in the cylindrical porous implant. In vivo, approximately 40% of the visible volume of the implants was degraded after 6 months and replaced by bone, showing the capacity to stimulate new bone formation. Histologically, ample bone ingrowth was observed. In contrast, empty defects were filled with fibrous tissue only, confirming the material’s osteoconductive capacity. It is concluded that this study provides proof that the 3D-printed brushite implants were able to promote new bone growth after 6 months’ implantation in a large animal model and that the new equine tuber coxae bone model that was used is a promising tool for bone regeneration studies.
Molecular characterization of Brucella ovis in Argentina
(Elsevier B.V., 2020-04-22) Alvarez, Lucía P.; Ruiz-Villalobos, Nazaret; Suárez-Esquivel, Marcela; Thomson, Nicholas; Marcellino, Romanela; Viquez Ruiz, Eunice; Robles, Carlos A.; Guzman-Verri, Caterina
La brucelosis en los carneros es causada por Brucella ovis o Brucella melitensis y se considera una de las enfermedades infecciosas más importantes de los machos en los países donde se crían ovejas. Caracterización molecular de Brucella spp. logrado por el análisis de número variable de múltiples locus de repeticiones en tándem (MLVA) es una herramienta poderosa para genotipar Brucella spp. Sin embargo, los datos sobre el genotipado de B. ovis son escasos. Así, el objetivo de este estudio fue caracterizar la diversidad molecular de cepas de campo de B. ovis en Argentina. Un total de 115 aislamientos de B. ovis de Argentina y Uruguay fueron genotipados usando MLVA-16 y analizados en conjunto con 14 B. ovis disponibles públicamente .genotipos de Brasil. El Poder Discriminatorio (D) fue 0,996 para MLVA-16 y 0,0998 para MLVA-8 y MLVA-11. El análisis de MLVA-16 reveló 100 genotipos diferentes, todos ellos novedosos, incluidos 90 únicos. No hubo correlación entre la distribución geográfica y el genotipo y los resultados mostraron una mayor diversidad dentro de las provincias que entre las provincias. El análisis de agrupamiento de las cepas de Argentina, Uruguay y Brasil reveló que los 129 aislamientos se agruparon en dos clados. El análisis de secuenciación del genoma completo de los 19 genomas de B. ovis disponibles en las bases de datos públicas, e incluyendo algunas de las cepas argentinas utilizadas en este estudio, reveló la agrupación de los aislados argentinos y una relación más estrecha con B. ovisde Nueva Zelanda y Australia. Este trabajo agrega nuevos datos al mal entendido mapa de distribución de genotipos a nivel regional y mundial para B. ovis y constituye el estudio más grande de genotipado molecular de B. ovis hasta ahora.

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