RESUMEN
BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.
Asunto(s)
Infecciones por Picornaviridae , Picornaviridae , ARN Viral , Enfermedades de los Porcinos , Animales , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Porcinos , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , ARN Viral/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Sensibilidad y Especificidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedad Vesicular Porcina/diagnóstico , Enfermedad Vesicular Porcina/virología , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Brasil , Reproducibilidad de los ResultadosRESUMEN
Leishmaniasis is a zoonotic disease with worldwide distribution. In the Americas, the causative agent of the visceral form is the protozoa Leishmania (Leishmania) infantum. Transmission to the host or vertebrate reservoir occurs through the bite of infected arthropod females like Lutzomyia longipalpis. The epidemiological connection between the infection in dogs and humans generate constant studies about the relationship between the parasite and the canine host, including the development of methods and tests for the detection and quantification ofLeishmania (L.) infantum. Both conventional PCR (cPCR) and quantitative PCR (qPCR) can be used in the diagnosis of the parasite. Dropet Digital PCR (ddPCR) is another useful tool. Knowing the parasite load and its relationship with the clinical signs of naturally infected dogs is useful in research development and for establishing treatments that reduce the transmission of the disease. In this study, thirty-nine clinical samples of spleen from dogs naturaly infected by L. infantum were collected after necropsy. Two molecular tools were used to quantify the parasite load (qPCR and ddPCR) and there was 100% agreement in the results of the them. The tools developed in this work are important for the detection of L. infantum in dogs and humans. Droplet Digital PCR does not require a standard curve and is easy to standardize. In such manner, this new tool can generate more in-depth information in the broad debate about parasitic loads and the pathogenesis of leishmaniasis.
RESUMEN
BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.
Asunto(s)
Leishmania infantum , Leishmaniasis Visceral , Humanos , Animales , Perros , Leishmania infantum/genética , Leishmaniasis Visceral/veterinaria , Brasil , ARN Polimerasa Dependiente del ARNRESUMEN
Burkholderia mallei is the causative agent of glanders, a zoonosis listed by the World Organization for Animal Health as of mandatory notification. In this work, a comparison of three qPCR protocols was made, two of them based on articles by other authors and one standardized in house, this last one aiming at a genomic region that does not exist in other species of the Burkholderia genus. All qPCRs showed high efficiency and good repeatability. However, reactions with Cq between 36 and 40 were considered suspicious and unreliable, requiring greater clinical criteria to analyze the results.
Asunto(s)
Burkholderia mallei , Muermo , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Burkholderia mallei/genética , Muermo/diagnóstico , Caballos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reproducibilidad de los ResultadosRESUMEN
Brucellosis is an infectious disease caused by bacteria of the genus Brucella, which affects domestic animals and is transmissible to humans. The objective of this study was to evaluate six methods of DNA extraction directly from bovine tissue to detect Brucella spp. The Cq values for all samples were above 30 and varied according to the extraction kit used, but four kits showed no statistical difference in sensitivity. This work demonstrates the importance of choosing the best extraction kit before validation of a molecular diagnostic technique.
Asunto(s)
Brucella/genética , Brucelosis/diagnóstico , Brucelosis/veterinaria , Enfermedades de los Bovinos/diagnóstico , ADN Bacteriano/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Animales Domésticos/microbiología , Brucella/clasificación , Brucella/aislamiento & purificación , Brucelosis/microbiología , Bovinos , Enfermedades de los Bovinos/microbiología , Humanos , Técnicas de Diagnóstico Molecular/veterinaria , Sensibilidad y EspecificidadRESUMEN
Bovine tuberculosis is a disease that is widely distributed around the world. Its causative agent, Mycobacterium bovis, has characteristics of a microorganism with clonal multiplication in populations with no evidence of genetic exchange between strains, and, consequently, a group of strains can be identified as descending from a common ancestor. The aim of this study was to investigate the clonal complexes of M. bovis isolated from samples of lesions suggestive of bovine tuberculosis collected from slaughterhouses in various states of Brazil between 2006 and 2012. Ninety samples were analyzed, and it was found that 14.4% belonged to the clonal complex European1 and 81.1% to the clonal complex European2, while 4.65% were not identified as any of the four known complexes.
Asunto(s)
Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Tuberculosis Bovina/epidemiología , Animales , Brasil/epidemiología , Bovinos , Evolución Clonal/genética , Mycobacterium bovis/aislamiento & purificación , Tuberculosis Bovina/microbiologíaRESUMEN
Swine are the only known hosts of swinepox virus (SWPV), the sole member of the genus Suipoxvirus, family Poxviridae. Rapid diagnosis is recommended for appropriate interventions because of the high morbidity associated with this virus. This study describes a real-time quantitative PCR (qPCR) assay for rapid detection and quantification of SWPV. The detection limit, repeatability, reproducibility, and specificity of this assay were determined. The efficiency was 96%, and the R2 value was 0.996. The detection limit was 1 fg or 10-0.5 TCID50/50 µL. Tests showed that the greatest source of error in the SWPV qPCR assay was variation between analysts rather than different qPCR kits or equipment. All nucleic acids from other viruses or samples collected from swine were negative in the specificity test. qPCR for SWPV is a new method with tested variables that allows main sources of error in laboratory diagnosis and viral quantification to be identified.
Asunto(s)
Infecciones por Poxviridae/diagnóstico , Suipoxvirus/genética , Enfermedades de los Porcinos/virología , Animales , ADN Viral/genética , Límite de Detección , Infecciones por Poxviridae/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa , Suipoxvirus/clasificación , Suipoxvirus/aislamiento & purificación , PorcinosRESUMEN
Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.
Asunto(s)
Enfermedades de los Bovinos/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Brasil/epidemiología , Búfalos/virología , Bovinos , Enfermedades de los Bovinos/epidemiología , Infecciones por Herpesviridae/epidemiología , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/clasificación , Herpesvirus Bovino 1/genéticaRESUMEN
Infection with ovine gammaherpesvirus 2 (OvHV-2) is generally asymptomatic in sheep; however, when it crosses the species barrier, it causes malignant catarrhal fever (MCF) in cattle. In the present study, we developed a real-time PCR assay and a droplet digital PCR assay and use both methods to study an outbreak caused by OvHV-2. Both PCR methods showed high sensitivity and specificity and were able to detect low copy numbers of OvHV-2 in sheep and cattle. The present study describes the first digital PCR quantification of OvHV-2 genome copies in samples collected from sheep and cattle.
Asunto(s)
Gammaherpesvirinae/genética , Fiebre Catarral Maligna/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de las Ovejas/virología , Animales , Bovinos , Variaciones en el Número de Copia de ADN , Brotes de Enfermedades , Genoma Viral , Fiebre Catarral Maligna/epidemiología , Sensibilidad y Especificidad , OvinosRESUMEN
BACKGROUND: The World Health Organization (WHO) has classified human zoonotic tuberculosis (TB) due to Mycobacterium bovis as a neglected issue in the developing world. In a recent cross-sectional study in Brazil, three of 189 TB patients presented with a coinfection of M. bovis and M. tuberculosis and were selected as cases for this study. OBJECTIVE: The aim was to evaluate risk factors (RF) for zoonotic TB in an urban area of Brazil in order to guide preventive programmes. METHODS: A matched case-control study was carried out nested within a cross-sectional study. For each of the three cases, 14 age- and sex-matched controls (TB due to M. tuberculosis) were selected. FINDINGS: Zoonotic potential exposures (ZE) and extrapulmonary TB (EPTB) were independently associated with zoonotic TB in multivariate analyses. CONCLUSIONS: ZE by occupation and consumption of raw milk and derivative products that place individuals in direct and indirect contact with animals and their excretions/secretions increase the risk for zoonotic TB in Brazil, especially among those with EPTB. Therefore, measures such as efficient control of bovine TB, distribution of pasteurised milk and its derivative products, and the diagnosis and monitoring of zoonotic TB in humans are essential steps, especially in developing countries where bovine TB is enzootic, and further studies are necessary.
Asunto(s)
Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/epidemiología , Tuberculosis/microbiología , Brasil/epidemiología , Estudios de Casos y Controles , Coinfección , Estudios Transversales , Humanos , Factores de Riesgo , Población UrbanaRESUMEN
The objective of this work is to describe the distribution of outbreaks of vaccinia virus (VACV), pseudocowpox virus (PCPV), and bovine papular stomatitis virus (BSPV) in Brazil. The Official Laboratory of the Brazilian Ministry of Agriculture received 89 samples from different locations in Brazil in 2015 and 2016 for diagnosis of vesicular and exanthematous disease. Poxvirus coinfections occurred in 11 out of 33 outbreaks, including the first reported triple infection by BPSV, PCPV, and VACV. This occurrence may be associated with the circulation of these viruses in Brazilian cattle.
Asunto(s)
Enfermedades de los Bovinos/virología , Coinfección/veterinaria , Brotes de Enfermedades/veterinaria , Infecciones por Poxviridae/veterinaria , Poxviridae/clasificación , Animales , Brasil/epidemiología , Bovinos , Coinfección/epidemiología , Coinfección/virología , Infecciones por Poxviridae/epidemiología , Infecciones por Poxviridae/virologíaRESUMEN
Although PPV has been described as a cellular contaminant, few recent studies about the presence of this virus in cell cultures, serum, and trypsin were found in the literature. The purpose of this study was to detect the presence of porcine parvovirus (PPV) by polymerase chain reaction (PCR) in cell cultures, serum, and trypsin used in official public laboratories of educational institutes and research centers. We tested samples of cell cultures (88), batches of trypsin (10), and fetal bovine serum (13) from different manufacturers. The PCR for beta-actin and GAPDH was used to evaluate the efficiency of DNA extraction from samples. The PPV DNA was detected in 52 of 88 (59.1%) cell culture samples. One in ten batches of trypsin tested for PPV DNA was positive. In no sample of fetal bovine serum, amplification of PPV DNA was observed. Positive samples were tested and confirmed by another analyst. In addition, all positive samples were sequenced. Our results indicate that regular PCR testing for PPV in cell cultures and their supplies is important.
Asunto(s)
Técnicas de Cultivo de Célula , ADN Viral/genética , Infecciones por Parvoviridae/genética , Parvovirus Porcino/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , Bovinos , Infecciones por Parvoviridae/diagnósticoRESUMEN
In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.
Asunto(s)
Coinfección/microbiología , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Adolescente , Adulto , Brasil/epidemiología , Coinfección/epidemiología , Estudios Transversales , ADN Bacteriano/análisis , Escolaridad , Femenino , Humanos , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Tuberculosis/diagnóstico , Tuberculosis/epidemiología , Población Urbana , Adulto JovenRESUMEN
Vesicular stomatitis caused by Alagoas vesiculovirus (VSAV) has generated disease outbreaks in Brazil, mainly in the northeast region. Phylogenetic studies divide the isolates into three distinct genotypes (A, B, and C). However, there is no description of how this genetic divergence reflects on the phenotype of VSAV isolates such as in vitro replication fitness. Therefore, the objective of this work was to evaluate the ability of three distinct genotypes of Brazilian isolates of VSAV to grow in different cell-culture lines (BHK-21, Vero, and NCI-H1299). Quantification of viral RNA was performed using RT-PCR digital droplet from supernatant of cell culture collected every 4 h for a period of 24 h of viral growth in three different cell lines (BHK-21, Vero, and NCI-H1299). It was observed that the genotype C isolate has the lowest replication efficiency among the three analyzed viruses, without major changes in the copies of viral RNA over the entire time of the study.
Asunto(s)
Estomatitis Vesicular , Vesiculovirus , Animales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Filogenia , Vesiculovirus/genética , ARN Viral/genéticaRESUMEN
The pseudocowpox virus (PCPV) is recognized for causing exanthematic lesions in cattle and humans. The diagnosis is important because it is a zoonosis and its clinical signs can be confused with foot-and-mouth disease, a high-impact bovine disease in livestock. The objective of this work is to validate a SYBR Green qPCR and a conventional PCR for virus detection in bovine samples. Detection limit tests, repeatability, reproducibility, sensitivity, and specificity were compared. When two analysts were compared, results demonstrated that training and pipetting influence the repeatability. The qPCR was more sensitive than conventional PCR but showed nonspecific reactions distinguishable by the melting curve. Both showed high repeatability and reproducibility.
Asunto(s)
Enfermedades de los Bovinos , Infecciones por Poxviridae , Animales , Bovinos , Enfermedades de los Bovinos/diagnóstico , Patología Molecular , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/veterinaria , Virus de la Seudoviruela de las Vacas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Malaria is an acute febrile disease caused by a protozoan of the genus Plasmodium. Light microscopy (LM) is the gold standard for the diagnosis of malaria. Despite this method being rapid and inexpensive, it has a low limit of detection, which hampers the identification of low parasitemia infections. By using multicopy targets and highly sensitive molecular techniques, it is possible to change this scenario. In this study, we evaluated the performance of droplet digital PCR (ddPCR) to detect Plasmodium DNA obtained from saliva samples (whole saliva and buccal swab) of 157 individuals exposed to malaria transmission from the Brazilian Amazon region. We used the highly sensitive ddPCR method with non-ribosomal multicopy targets for Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). There was good concordance between the quantitative real-time PCR (qPCR) results from the saliva and blood, except for mixed-species infections. The sensitivity of qPCR was 93% for blood, 77% for saliva, and 47% for swabs. Parasite DNA was not detected in saliva samples in low-density infections compared with the detection in blood samples. ddPCR showed increased sensitivity for detecting Plasmodium in the blood and swabs (99% in blood, 73% in saliva, and 59% in swabs). Notably, ddPCR detected more mixed infections in the blood (15%), saliva (9%), and swabs (18%) than qPCR. Our data showed that the differences between ddPCR and qPCR were the result of a higher number of P. falciparum infections detected by ddPCR. Overall, there was a moderate correlation between parasite densities estimated by the different methods in the blood. Our findings highlight the possibility of using non-invasive sample collection methods for malaria diagnosis by targeting multicopy sequences combined with highly sensitive molecular methods.
RESUMEN
The vesicular stomatitis virus belongs to the Rhabdoviridae family, genus Vesiculovirus. Four species (New Jersey, Indiana, Cocal, and Alagoas) are responsible for disease outbreaks in Western Hemisphere countries. In Brazil, the Alagoas virus is responsible for the main outbreaks of the disease, mainly in the states of the Northeast, Midwest, and Southeast regions of the country. The present study aimed to perform the genetic characterization of 41 vesicular stomatitis virus samples. RNA was extracted using Trizol and used to amplify part of gene P. Amplicons were sequenced using the Sanger method. The phylogenetic trees generated showed that Alagoas vesiculoviruses were positioned into three groups: group A formed by the first virus isolate; group B by isolates from states in the Northeast region; and group C by isolates from the states of Bahia, Goiás, and Tocantins. Their divergence to date has generated the formation of two genotypes evolving independently in regions that until the present study had little geographic overlap.
Asunto(s)
Estomatitis Vesicular , Animales , Brasil/epidemiología , Filogenia , Virus de la Estomatitis Vesicular Indiana/genética , Vesiculovirus/genéticaRESUMEN
BACKGROUND: Minas artisanal cheese (MAC) from the Serro region is a Brazilian intangible cultural heritage. Produced from raw milk, it may carry zoonotic pathogens such as Brucella. This study included a randomized survey for the prevalence of Brucella-positive MAC and its associated factors. METHODS: MAC samples (n=55), each one from a different rural family-based cheese-processing agroindustry, were analysed for Brucella by direct polymerase chain reaction (PCR) species-specific DNA detection and cultivation-based approaches. RESULTS: Among 55 MACs that were analysed, we found 17 Brucella DNA-positive samples (30.9% [95% confidence interval {CI} 18.7 to 43.1]) by PCR and, for the first time, from one MAC (1.8% [95% CI 0.5 to 9.7]), viable Brucella abortus was recovered by cultivation. Higher values for two variables, the number of lactating cows per herd (p=0.043) and daily milk production per herd (p=0.043), were each associated with Brucella-positive MAC, which concentrated in three high-risk and one low-risk spatial clusters. CONCLUSIONS: MAC may be a source of Brucella for humans, since the positive samples were from batches that were sold by cheesemakers. This should be of concern and encourage cooperation between the health and agriculture sectors in order to mitigate this public health risk through One Health integrated approaches.
Asunto(s)
Brucella , Queso , Salud Única , Femenino , Bovinos , Humanos , Animales , Queso/análisis , Brasil/epidemiología , Leche , Prevalencia , Lactancia , Factores de RiesgoRESUMEN
This article describes the recurrence of outbreaks of Vesicular Stomatitis in the State of Maranhão, Brazil. The procedures for treating the outbreak of vesicular disease, sample collection, laboratory tests performed, and the results obtained were described. The clinical signs and observed injuries have been described. The sera showed antibodies that cross-react between the Vesiculovirus Indiana, Cocal, and Alagoas. The serological profile shows the presence of high antibody titers for Alagoas vesiculovirus in cattle, swine, and horses. Higher antibody titers indicate the viral serotype present in the outbreak. The genetic sequencing of the isolates confirmed the presence of Alagoas vesiculovirus, which grouped with the virus isolated in 2013 from cattle from the State of Maranhão.
Asunto(s)
Estomatitis Vesicular , Vesiculovirus , Animales , Brasil/epidemiología , Bovinos , Brotes de Enfermedades/veterinaria , Caballos , Serogrupo , Porcinos , Estomatitis Vesicular/epidemiología , Vesiculovirus/genéticaRESUMEN
Bovine leukemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus. BLV infects cattle worldwide and is responsible for significant economic losses. The objective of this study was to validate real-time quantitative PCR (qPCR) for the detection of BLV. After identification of the most efficient qPCR, the limits of detection, repeatability, and reproducibility were determined. The results indicate that qPCR can be easily reproduced between laboratories with high sensitivity. The test variation was low in samples from lesions suggestive of bovine leukosis or whole blood.