Browsing by Author "Vallecillo Maza, Antonio Javier"

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Actualización de la seroepidemiología de toxoplasma gondii en gatos de la ciudad de Cuenca
(2016) Bojorque Pazmiño, Marcia Priscila; Vallecillo Maza, Antonio Javier
Toxoplasmosis is a cosmopolitan disease which pathological agent is a protozoon kwon as toxoplasma gondii, responsible of a variety of clinic signology, morbility and mortality in the different animal species it infects. It is intimately related with the acquisition and possession of domestic cats, also considered synonymous of miscarriage and pregnancy difficulties in the pregnant woman, placing it under the scrutiny of public health programs. On 1994 in Cuenca city a study was made for the detection of antibodies against Toxoplasma gondii in felines, resulting in 44.5% of positive cats. Over time cats have became the ideal mascot due to their space, behavior and feeding characteristics. Even more for a population that is increasing every day and prefers because of its life style easy care animals. On the basis of the said before and on the significant development on the veterinarian professional area dedicated to minor species, we decide to carry out an investigative project which main objective is the actualization of data related to toxoplasma gondii and cats in the city. The lab test used for this investigation differs from the previous essay, blood samples obtained from cats where analyzed by indirect immunofluorescence method because of its specificity and sensitivity. Results found will allow us to have an updated vision of the serum prevalence of toxoplasmosis. Data obtain on this investigation are minor to the ones obtain two decades ago, due to the increase of awareness acquired on handling, feeding and hygiene by the owners of feline pets which is determining for the development of the disease, also a bigger influx to veterinary centers for before and after information of a recent acquisition or adoption of a cat.
Aplicación de un ensayo de PCR convencional múltiple para la detección y diferenciación de cepas de campo y cepas vacunales S19 y RB51 de Brucella abortus en muestras biológicas de bovinos
(2017) Aucancela Yunganaula, María Elena; Vallecillo Maza, Antonio Javier
Bovine Brucellosis is an infectious-transmissible disease of global distribution, caused by a bacterium of the genus Brucella. This disease is characterized by abortions in the last third of gestation and reduced fertility, representing a serious economic and sanitary problem. The objective of this work was to evaluate a multiplex PCR assay for the diagnosis of Brucella abortus infection in milk and lymph node samples from seropositive and seronegative bovines distinguishing field strains from vaccine strains S19 and RB51. For serology (Rose Bengal) blood samples were collected from 200 animals, of which 82 cows were in milk production from which milk samples for PCR were taken, plus 69 samples of lymph node tissue (right and / or left prescapular and supramammary). Eight percent of the samples were found to be seropositive by rose bengal test, whereas only 1.2% of the samples resulted positive using the multiplex PCR, corresponding to a milk sample that presented a 456 bp amplification band, coincident with the expected amplicon length of the S19 vaccine strain. The remaining milk samples and in the tissue samples produced several thin bands of different sizes, being nonspecific amplifications, so it is concluded that the multiplex PCR evaluated in this study was not applicable for diagnosis with the primers used.
Bovine neosporosis in dairy cattle from the southern highlands of Ecuador
(2020) Maldonado Rivera, Jaime Eduardo; Vallecillo Maza, Antonio Javier; Perez Meneses, Cinthya Lizbeth; Cirone, Karina; Dorsch, Matías; Morrell, Eleonora Lidia; Scioli, Valeria; Hecker, Yanina Paola; Fiorani, Franco; Cantón, Germán José; Moore, Dadín
The aim of this study was to describe bovine neosporosis in dairy cattle from the Sierra region, Ecuador. A case-control study was performed on 841 dairy cattle from 5 dairy herds. The overall seroprevalence was 23.4% having significant association between abortion and seropositivity (p < .05). Additionally, 46 fetuses were recovered from a local slaughterhouse to evaluate the frequency of vertical transmission. Seventeen and 3 fetuses were positive by PCR and had compatible histopathological lesions, respectively. N. caninum infection must be considered as a relevant cause of reproductive losses in Ecuador.
Características génicas de las poblaciones de bovinos criollos americanos en base a microsatélites
(2019) Guevara Viera, Guillermo Emilio; Vallecillo Maza, Antonio Javier; Méndez Álvarez, María Silvana; Soria Parra, Manuel Elías; Palacios Ordóñez, Teofilo Estuardo; Andrade Guzmán, Omar Santiago; Bustamante Ordóñez, Jorge Gualberto; Pesántez Pacheco, José Luis; Vázquez Mosquera, Juan Mesías
The objective of this study is to analyze the major gene parameters found in different genotypes of cattle of Iberian origin in the Americas. The work was based on the review of different results of the genetic markers analyzed in blood of Creole cattle incountries of North America until cattle in Patagonia in the southern part of South America. Presents results of the markers most used and recommended for ca ttle. Population genetic parameters analyzed were: total number of alleles (Na), by microsatellite marker allele 189 frequencies), the factors observed (Ho) and expected (He), the coefficients of inbreeding (Fis), polymorphic information content) CIP) and compliance with the assumption of Hardy-Weinberg equilibrium. The percentage of variance between populations and the (Fst) is also reported. 20 papers on microsatellites in cattle, analysed 19 of them with informa tion from Creoles of America. Highlights a number of alleles locus of 4 to 13, higher by more than 0.6 heterozygosity and polymorphic information content high. Creole ca ttle have very little variation among their populations, the distance that separates them is not very marked, but they have a Kingdom with many alleles at the loci analyzed and their heterozygosity and polymorphism is high of them with information
Caracterización molecular de las cepas de Parvovirus canino tipo 2 en perros de la ciudad de Cuenca
(2020-01-07) Clavijo Izquierdo, Verónica Andrea; Vallecillo Maza, Antonio Javier
The canine parvovirus is a contagious disease caused by canine parvovirus type 2 (CPV-2). It especially affects puppies under six months and adult dogs without immune protection. The signology includes lethargy, anorexia, fever, vomiting and diarrhea; if the animal doesn’t get support treatment, the illness can be lethal. The original strain CPV-2 has been completely replaced by three variants (CPV-2a, CPV-2b, CPV-2c), and this evolution has occurred in a relatively short time. However, there is controversy regarding how virulent these strains are, as well as the level of protection of the current vaccines. The aim of this study was to identify the different strains of CPV-2 in dogs with parvovirosis compatible signs in the city of Cuenca. For this, 80 feces samples were collected, from which those that did not generated a specific amplicon of dog (Process control) were discarded. Subsequently, the disease diagnostic PCR was performed and 25 positive samples were selected and sent to sequencing. From these samples, it was possible to obtain the genetic sequence of 21. The 47,61% belonged to strain CPV-2a, followed by strain CPV-2c with 38.1%, and finally the strain CPV-2b with 14,29%. According to what has been published, these strains suffer prevalence changes and can generate subvariants of itselfs over the time and in different geographic locations.
Comparación de alelos de receptores para FSH y LH identificados mediante el ensayo PCR-RFLP en bovinos Holstein y Criollo de la provincia del Azuay
(Universidad de Cuenca, 2021-04-14) Cuzco Paida, Xavier Alejandro; Vallecillo Maza, Antonio Javier
Variability in reproductive genes, such as those that encode receptors for follicle-stimulating (FSHR) and luteinizing (LHR) hormones, has decreased in recent decades, both in specialized dairy cattle and in creole cattle. Curiously, in this last breed a high variability is suggested, important for genetic improvement and its conservation. The objective of the study was to contrast the frequency of the FSHR and LHR genes in the Holstein and Creole bovine populations of the Azuay province by means of an assay based on the polymerase chain reaction and the identification of restriction fragments of polymorphic length (PCR- RFLP) from DNA of both races. Three genotypes were identified: CC; CG and GG for FSHR, and only one (GG) for LHR, the entire population being homozygous for this locus. For FSHR, 56% of the individuals were CC homozygous, with a higher percentage of these in Holstein cattle (67%); while, in the Criollo cattle, 55% were heterozygous, being the C allele predominant in both breeds. Greater heterozygosity was observed for the FSHR locus, in Criollo cattle with observed heterozygosity (Ho) (0.5517) higher than expected (He) (0.3995); while Holstein cattle presented moderate heterozygosity; Furthermore, in both breeds there was no evidence of consanguinity. Both the genetic differentiation and the distances between these two races were small. The total population was found in genetic equilibrium for FSHR, these results allowed characterizing the study population as non-inbred, with the same allelic frequencies and with greater variability in the Creole race for this locus.
Construcción de un vector de expresión bacteriano para la producción y purificación de proteínas recombinantes mediante la actividad de sortasa A de Staphylococcus aureus
(Universidad de Cuenca, 2022-04-13) Cárdenas Cordero, Janneth Fernanda; Vallecillo Maza, Antonio Javier
Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1
(CAMBRIDGE UNIVERSITY PRESS, 2016-01-01) Vallecillo Maza, Antonio Javier
Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ?25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.
Determinación de la diversidad genética y caracterización de genes de patogenicidad en aislados de Escherichia coli en pollos de engorde (Gallus gallus domesticus), Azuay, Ecuador
(2023) Astudillo Riera, Fabián Manuel; Vallecillo Maza, Antonio Javier; Perez Pintado, Ana Cecilia; Rivera Pirela, Sergio Emiro
In the poultry industry there are different types of pathogenic and non-pathogenic Escherichia coli, depending on the virulence associated genes (VAG) present in the bacteria. However, there are no reports for the area on the identification of E. coli VAG in broiler chickens especially in Ecuadorian andean communities. The main objective of this study was to identify the genetic diversity and the presence of VAG in E. coli strains. In this study, E. coli VAG were characterized in the Azuay Province (Ecuador) using isolates from 30 bacteriological cultures from chickens with clinical signs of colibacillosis. The PCR technique was used to detect the uspA gene (specific from E. coli) and also the GAV of adhesins (fimC), exotoxins (cvaA) and iron uptake-transport systems (iucD; chuA; fyuA) in each bacteria isolate. Pathogenic E. coli were molecularly typed by evaluating polytrinucleotide (GTG)5 sequences. The 83.33 % of the cultures presented the uspA gene from E. coli. The frequencies of positive VAG were 48 % for the chuA gene, 20 % for the cvaA gene, 84 % for the fimC gene, 36 % for the fyuA gene and 56 % for the iucD gene. Sequence evaluation (GTG)5 revealed two main phylogenetic groups of E. coli most of which carried at least one VAG gene. These results contribute to a more precise diagnosis of colibacillosis, as well as to is control and treatment by poultry farmers.
Diagnóstico molecular de la infección gastrointestinal de neospora caninum en caninos
(2018) Barbecho Quinche, Pedro Bernini; Vallecillo Maza, Antonio Javier
Neosporosis in a parasitic infection caused by the protozoan Neospora caninum, the same that was first observed in 1984; it definitive host the mammals of the family of dogs, coyotes, dingos, etc., and their intermediary hosts are the bovine family, sheeps, goats, etc. This parasite is characterized by causing a long variety of neurological symptoms in infected animals, such as ataxia, muscular atrophy and muscle pain in dogs; while in cattle they can cause abortions, reabsorption or fetal mummification, among other pathologies. This research work is a quantitative cross - sectional type, since it was proposed with the purpose of knowing the sensitivity and specificity diagnostic of this parasite in dogs of the rural area of the canton Cuenca in the year 2017, for this it was done a test of feces through the diagnostic methods of Sheather's Sugar and CRP to 100 randomly chosen dogs, to later compare the results obtained by these two diagnostic techniques and present statistical analyzes using simple and composite tables, as well as statistical graphs. An incidence of 6 % (6 cases) was obtained as a result of the coproparasitary examination and an incidence of 1.06 % (1 case) using the PCR technique, however, there was no statistical relationship between these two variables, because the coproparasitary examin has a low sensitivity and specificity of in relation to the PCR test.
Estimación combinada de número alelos, heterocigosidad esperada y observada en microsatélites de bovinos mediante metaanálisis
(2019) Guevara Viera, Guillermo Emilio; Soria Parra, Manuel Elías; Méndez Álvarez, María Silvana; Vallecillo Maza, Antonio Javier
Evaluación de la aplicación de dos ensayos moleculares (PCR y LAMP) para la identificación de material genético de Neospora caninum en sangre de Canis lupus familiaris (perro)
(2019) Abril Vásquez, Marcela Estefanía; Siguenza Guaman, Nidia Mirella; Vallecillo Maza, Antonio Javier
Neospora caninum is an Apicomplexa parasite, the etiological agent of neosporosis, a disease considered of great importance in the productive and reproductive losses of bovines and neuromuscular damages in dogs, with a worldwide presence. This protozoan parasite is kept in the environment by a heteroxen cycle that involves as a definitive host the dog (Canis familiaris or Canis lupus familiaris) and other animals of the Canis genus such as Coyote (Canis latrans) or Dingo (Canis lupus dingo) and it has several intermediate hosts, the most important being cattle. The objective of this study was to compare two molecular techniques, PCR and LAMP for the identification of genetic material of N. caninum, in peripheral blood samples obtained from dogs, without distinction of sex, age or breed, from the livestock areas adjacent to the city of Cuenca that live with bovines and are exposed to the consumption of biological waste of these animals, this being the population most susceptible to develop damage to their health from this parasite and less chance of cure. For the above, blood samples were taken from 100 dogs to subsequently extract the total DNA from each of them and apply both molecular methods, the results obtained showed two positive samples to N. caninum in PCR and 3 positive in LAMP, in the statistical analysis applied with Chi-square and FFisher tests, it was found that there is no statistical difference between the results obtained when applying these molecular methods, that is, this study suggests that both tests are equally efficient to identify genetic material from N. caninum in the peripheral blood of dogs. Finally, both techniques are considered highly specific and sensitive, both analytical and diagnostic compared to the serological tests applicable for the diagnosis of this disease, however the LAMP assay is more feasible to be applied in limited resource conditions since it does not require expensive equipment for its realization.
Evaluación de la proteína quimérica dD(SpA)-p6-dC1(SpG)-ApEcoli para la detección de anticuerpos
(Universidad de Cuenca, 2025-02-11) Orellana Benalcázar, Karla Mercedes; Sacta León, Carmen Rocío; Vallecillo Maza, Antonio Javier
Diagnosis of infectious diseases in veterinary medicine is essential for the adequate and timely management of diseases in animals. To achieve this objective, the availability of diagnostic tests is necessary; however, currently these tools must be imported. Therefore, this study presents the functional evaluation of the chimeric protein dD(SpA)-p6-dC1(SpG)- ApEcoli, which allows the detection of canine (Canis lupus familiaris) and bovine (Bos taurus) antibodies in assays such as indirect ELISA and Western blot. The protein combines the D domain of protein A (Staphylococcus aureus), the C1 domain of protein G (Streptococcus spp.), and Alkaline phosphatase from Escherichia coli. The production of the protein was carried out in the bacterial strain E. coli Rosetta 2 (DE3), using the plasmid pET28-PAp6PG-APEcoli-HT. After induction with IPTG and purification by immobilized metal affinity chromatography (IMAC), the presence of the protein and enzymatic activity were evident indirect ELISA, Western blot and Dot blot assays demonstrated that the dD(SpA)-p6- dC1(SpG)-ApEcoli protein recognizes antibodies from seropositive canines and bovines against antigens such as the VP2 protein of Carnivore protoparvovirus 1 and the p24 of Bovine leukemia virus. This development is relevant for Ecuador, where there is no production of local inputs for the development of diagnostic tests for diseases of veterinary interest. The protein obtained could reduce costs and improve access to accurate diagnoses for a wide range of animal species, contributing to the control of infectious diseases.
Evaluación de una ADN polimerasa Bst_LF de producción local en ensayos LAMP para la detección de Neospora caninum en muestras de bovinos (Bos taurus)
(Universidad de Cuenca, 2023-08-02) Sigüenza Jiménez, Estefanía Tatiana; Vallecillo Maza, Antonio Javier
Neospora caninum is an intracellular parasite of great importance in cattle breeding due to the great economic losses, both productive and reproductive. In our country, molecular techniques applicable to diagnosis are not available to most farmers due to the costs and time required to obtain the results, which prevents an accurate diagnosis of the disease. The objective of this study is to evaluate the use of a DNA polymerase Bst_LF enzyme produced in the Molecular Biology Laboratory of the Faculty of Agropecuary Sciences of the University of Cuenca in a LAMP assay, for the detection of N. caninum genetic material in biological samples of bovine (Bos taurus), using a commercial enzyme as a reference. We used 50 fetal brain tissue samples and 50 milk samples. The results obtained in the milk samples with the two enzymes were 100 % negative, therefore it was not possible to detect DNA of the pathogen. In the brain samples, 40% of positive samples were identified with the commercial enzyme and 26% of positive samples with the local enzyme, there being a numerical difference but no significant differences with McNemar's analysis. With McNemar's statistical test there were no significant differences between both enzymes (p = 0.0654), therefore the local enzyme Bst_LF shows the same capacity for the detection of N. caninum genetic material in bovine fetal brain tissue compared to that of the commercial enzyme Bsm.
Evaluación del antígeno ESAT6-CFP10 recombinante de Mycobacterium tuberculosis para la detección de anticuerpos en primates no humanos potencialmente expuestos a tuberculosis
(Universidad de Cuenca, 2025-09-03) Lema Segovia, Geovanna Daniela; Vele Criollo, Daniela Belén; Vallecillo Maza, Antonio Javier
Mycobacterium tuberculosis complex is made up of bacterial species belonging to the Mycobacteriacea family, most of which cause tuberculosis, a zoonotic disease considered of public health importance. These bacterial pathogens are considered Gram-positive, non-sporulating, and capable of intracellular residence. In non-human primates, the species M. tuberculosis, M. bovis, and additionally M. avium have been identified. The main infection occurs through the inhalation of aerosols expelled from the respiratory tract of both animals and humans who have been infected with the active disease. Since these are wildlife, their current status in these species is unknown. The present study aimed to evaluate ESAT-6 and CFP-10 antigens integrated into a fusipon protein, for the detection of anti-M. tuberculosis in Capuchino monkeys (Cebus capucinus) and Chorongo (Lagothrix poeppigii) potentially exposed to this disease, for which a total of 14 blood samples were collected (10 from Capuchin monkeys and 4 from Chorongo) residing in a zoo-shelter in the city of Cuenca, these samples were analyzed using Western blot and indirect ELISA diagnostic techniques using the recombinant antigen HT-ESAT6-CFP10. The results obtained show that, of the 14 analyzed sera, no reactivity to the M. tuberculosis HT-ESAT6-CFP10 antigens was evident in the Western blot assay. However, the in-house indirect ELISA showed signs of reactivity, which can be assumed to be suspected cases. Finally, no differences were found in the application of either of the two diagnostic methods for the identification of tuberculosis in non-human primates.
Evaluación del reconocimiento del antígeno IalB de Bartonella hanselae por anticuerpos de caninos (Canis lupus familiaris)
(Universidad de Cuenca, 2025-09-30) Celi Cruz, Jorge Luis; Corte Suqui, Jonathan Fabricio; Vallecillo Maza, Antonio Javier
Bartonella henselae (B. henselae), a Gram-negative bacterium responsible for bartonellosis, is a chronic disease that affects both animals and humans. In this context, cats (Felis catus domesticus) are the main carriers, and the disease is transmitted through their fleas (Ctenocephalides felis). In dogs, infection with this bacterium often does not induce the appearance of obvious clinical signs, which complicates its diagnosis at points of care. In addition, there are few diagnostic tools available to identify chronically infected animals. A possible indicator for detecting this infection in dogs (Canis lupus familiaris) is the IalB antigen, a protein found in the outer membrane of B. henselae. Therefore, the objective of this study was to evaluate the possible recognition of IalB by antibodies in dogs potentially exposed to B. henselae in the city of Cuenca, Ecuador. To achieve this objective, an indirect enzyme-linked immunosorbent assay (ELISA) was performed, using the recombinant IalB protein as the antigen together with a modified version of the same by adding a polystyrene-binding peptide. This assay was carried out on two types of plates. In the indirect ELISA assay, 92 canine sera were evaluated, in which the presence of fleas had been reported in a previous three-month period. MultiSorp and MaxiSorp plates were used for the analysis, and statistical tests were applied to compare reactivity. The Friedman test showed that, in the MultiSorp plates, there were no significant differences between the two antigens, indicating equivalent reactivity. However, in the MaxiSorp plates, lower antibody binding was observed. In addition, a high correlation between IalB and IalB-PoBP was evident in MultiSorp (ρ = 0.87; p < 0.0001), while a moderate correlation was observed in MaxiSorp (ρ = 0.65), suggesting that fusion to the peptide does not substantially alter recognition. Finally, no correlation was found between the presence of fleas and antibody detection (ρ ≈ 0.0; p > 0.7), which could be attributed to limitations in risk assessment based on medical history and physical examination.
Evaluación del uso de dos proteínas recombinantes de Neospora caninum para la detección de anticuerpos en perros (Canis lupus familiaris)
(2020-03-12) Pacheco Pacheco, Elena Estefanía; León Lliguicota, Magaly Tatiana; Vallecillo Maza, Antonio Javier
Neosporosis is a parasitic disease caused by Neospora caninum, whose definitive host is the dog (Canis lupus familiaris), this can cause neuromuscular disorders and in some cases dermatitis, among other complications. Besides, cattles as intermediate hosts, have abortions and neonatal mortality. The objective of this study was to evaluate the application of the NcSAG4 and NcGRA7 recombinant proteins of Neospora caninum, in ELISA and Western blot assays to identify the presence of antibodies in dogs in contact with cattle and therefore at risk of ingesting their biological remains (aborted fetuses and placentas) infected with the parasite. ELISA and Western Blot assays using recombinant proteins could offer an effective immunodiagnostic tool for the identification of dogs exposed to Neospora caninum. For this, blood sera obtained from 100 domestic canines were used, regardless of age, race and sex, residing in contact with cattle. The results obtained showed an animal with the presence of antibodies both for the NcGRA7 recombinant protein (ELISA and Western blot assays) and for NcSAG4 (Western blot assay); three animals recognized the NcGRA7 antigen (two in ELISA and the third in ELISA and Western blot); and two against NcSAG4 (Western blot). In the statistical analysis with the Cochran Q Test it was found that there is no statistical difference when applying these serological methods, that is to say that both techniques using the two recombinant proteins behave similarly for the detection of antibodies.
Evaluación post mortem de la diseminación de las variantes del Protoparvovirus carnívoro 1 en perros afectados por parvovirosis canina
(Universidad de Cuenca, 2023-10-12) Morocho Sandoval, Silvia Jimena; Pacheco Atariguana, José Luis; Vallecillo Maza, Antonio Javier
Infection in dogs (Canis lupus familiaris) caused by Protoparvovirus carnivore 1 (PPVC-1) and its antigenic variants (2a, 2b and 2c) can spread through all tissues and manifest gastroenteric, cardiac and nervous. This is due to the tropism of the virus for mitotically active cells that favor its replication. With this background, the present study aimed to identify the presence of PPVC-1 genetic material and to evaluate the frequency of antigenic variants in the central nervous system and cardiac tissues of dogs that died with a diagnosis of canine Parvovirosis. A total of 108 samples were collected; rectal swabs (n = 27), intestinal (n = 27), cardiac (n = 27) and nervous (n = 27) tissue fragments. The total DNA obtained from the samples was subjected to two PCR assays, one as a process control and the other for identification of PPVC-1 genetic material. It was possible to amplify PPVC-1 genetic material in 87.5% of processed swabs, confirming infection in 21 dogs. Infection was also identified in heart and brain tissues, but not in intestine. Twenty nervous tissue samples (74.1%) and nine cardiac tissue samples (42.9%) were positive for PPVC-1 from which 20 amplicons were selected for sequencing. From sequence analysis, the PVC-2a antigenic variant was successfully identified in 100 % of amplicons of nervous (n = 16) and cardiac (n = 4) origin.
Generación del antígeno IalB de Bartonella henselae y una versión modificada por la adición de un péptido con afinidad al poliestireno
(Universidad de Cuenca, 2024-08-05) Encalada Ochoa, Edgar Vinicio; Orellana Benenaula, Juan Diego; Vallecillo Maza, Antonio Javier
Bartonellosis is a chronic and inapparent disease caused by Gram-negative bacteria of the Bartonella genus. Bartonella species are characterized by affecting multiple mammals, such as felines, canines, ruminants, rodents, humans, among other hosts. Domestic felines (Felis catus domesticus) act as reservoirs for Bartonella henselae; they can transmit the infection through different blood-sucking vectors, such as fleas, ticks, lice, mites, among others. Due to the absence of a sufficiently reliable indirect diagnostic test and the high frequency of infection reported in felines, the generation of the invasion-associated locus protein B (IalB), a protein located in the outer membrane of Bartonella henselae, was proposed and It is involved in intraerythrocytic adhesion and invasion. To obtain the coding sequence of IalB, assembly was carried out by the polymerase chain reaction (PCR)-thermodynamically balanced inside-out (TBIO) method. For the expression of IalB and an IalB-PoBP variant (Polystyrene binding peptide (PoBP) added to the carboxyl end), the Escherichia coli Rosetta 2 (DE3) strain was used; both antigens were purified by immobilized metal affinity chromatography (IMAC), having a yield of approximately 4,50 mg of pure protein per liter for IalB and 1,39 mg of protein for the case of IalB-PoBP. With both antigenic proteins obtained, an indirect ELISA test was carried out in order to evaluate the recognition by antibodies of domestic felines, obtaining similar results for both antigens, which should be evaluated with sera from animals previously identified as infected by Bartonella henselae
Identificación de actividad enzimática de fosfatasa en muestras de agua y lodo de la planta de tratamiento de aguas residuales de Ucubamba de la ciudad de Cuenca
(2017-10-26) Villegas Lituma, Tania Carina; Vallecillo Maza, Antonio Javier
The industrial, agricultural, pharmaceutical and domestic operations are sources of pollution to natural environments (soil, water, air) with heavy metals. This contamination produces loss of biodiversity, decrease of socio-economic activities and increase in diseases. Different studies have reported that phosphatase enzymes can remove heavy metals, so, this research looked for proteins with phosphatase enzymatic activity from wastewater and sewage of the Wastewater Treatment Plant of Ucubamba in the Cuenca city. The histochemical screening of metagenomics libraries built from PCR products of acid phosphatase (PhoN) cloned in the pET302/NT-His vector, expressed in E.coli BL21 (DE3) Start were carried out. For being identified phosphatase activity used an indicator medium LB-agar supplemented with phenolphthalein diphosphate and methyl green. With the above was achieved detecting enzymatic phosphatase activity in all the samples of wastewater and sewage, being more frequent in clones from wastewater de maturation lagoons.