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Lara Borges Keid - One of the best experts on this subject based on the ideXlab platform.
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MALDI-TOF MS and genomic analysis can make the difference in the clarification of Canine Brucellosis outbreaks
Scientific Reports, 2020Co-Authors: David Attuy Vey Da Silva, Lara Borges Keid, Rodrigo Martins Soares, Julia Teresa Ribeiro De Lima, Holger Brendebach, Josephine Grützke, Ralf Dieckmann, Dirk Hofreuter, Sascha Al DahoukAbstract:Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine Brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of Brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with Canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of Canine Brucellosis.
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Genome Sequences of Five Brucella canis Strains Isolated from Different Countries throughout the World.
Microbiology resource announcements, 2018Co-Authors: Guillaume Girault, Lara Borges Keid, Jane Megid, Acácia Ferreira Vicente, Yannick Corde, Mateus De Souza Ribeiro Mioni, Maryne Jay, Virginie MickAbstract:ABSTRACT Canine Brucellosis is a major underestimated zoonosis that remains endemic in many areas of the world. A recent phylogeographic investigation including 53 Brucella canis field isolates revealed the existence of two major lineages worldwide. Here, we report genome sequencing of 5 representative isolates of different clades identified in this study.
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New insights into phylogeography of worldwide Brucella canis isolates by comparative genomics-based approaches: focus on Brazil
BMC genomics, 2018Co-Authors: Acácia Ferreira Vicente, Lara Borges Keid, Jane Megid, Guillaume Girault, Yannick Corde, Mateus De Souza Ribeiro Mioni, Maryne Jay, Virginie MickAbstract:Background Canine Brucellosis, due to Brucella canis, is a worldwide zoonosis that remains endemic in South America, including Brazil. Implementation of powerful whole-genome sequencing approaches allowed exploring the Brucella genus considered as monomorphic, with, to date, more than 500 genomes available in public databases. Nevertheless, with under-representation of B. canis genomes −only twenty complete or draft genomes−, lack of knowledge about this species is still considerable. This report describes a comparative genomics-based phylogeographic investigation of 53 B. canis strains, including 28 isolates paired-end sequenced in this work.
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Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for Canine Brucellosis diagnosis
Molecular and cellular probes, 2018Co-Authors: Maria Cryskely Agra Batinga, Rodrigo Martins Soares, Julia Teresa Ribeiro De Lima, Fábio Gregori, J. A. Diniz, Kerstin Muner, Trícia Maria Ferreira De Sousa Oliveira, Helena Ferreira, Lara Borges KeidAbstract:Canine Brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of Brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for Canine Brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of Canine Brucellosis.
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Evaluation of an Immunochromatographic Test to the Diagnosis of Canine Brucellosis Caused by Brucella canis.
Reproduction in domestic animals = Zuchthygiene, 2015Co-Authors: Lara Borges Keid, J. A. Diniz, Trícia Maria Ferreira De Sousa Oliveira, Helena Ferreira, Rodrigo Martins SoaresAbstract:This study evaluated the performance of an immunochromatographic test (ICT) for the diagnosis of Canine Brucellosis caused by Brucella canis, comparing its results with that of the rapid slide agglutination test with and without the use of 2-mercaptoethanol and the agar gel immunodiffusion test (AGID). The microbiological culture, PCR and clinical examination were used as reference. According to the results obtained in clinical examination, blood culture, culture of semen and vaginal swab and PCR in blood, semen and vaginal swab, a total of 102 dogs were divided into three groups: B. canis-infected dogs (Group 1), B. canis-non-infected dogs (Group 2) and dogs with suspected Brucellosis (Group 3). The diagnostic sensitivity of RSAT, 2ME-RSAT, AGID and ICT in Group 1 was, respectively, 75%, 37.5%, 27.8% and 89.58%. The diagnostic specificity of RSAT, 2ME-RSAT, AGID and ICT in Group 2 was, respectively, 91%, 100%, 100%, and 100%. In dogs with suspected Brucellosis, 9.67% were RSAT positive, none was positive by 2ME-RSAT, 3.22% were AGID positive and 6.45% were ICT positive. The main drawback concerning Canine Brucellosis diagnosis is the lack of a highly sensitive serological assay to be used as a screening test to the rapid identification of infected animals. The ICT showed a high diagnostic specificity and a diagnostic sensitivity value greater than that observed in the RSAT, 2ME-RSAT and AGID. However, 10.41% of infected dogs had negative results by ICT. These dogs were positive by microbiological culture and/or PCR, indicating active infection and consequently a higher potential of spreading Brucella. Although rapid and simple to perform, the ICT lacked sensitivity to be used as a screening test.
M M Wanke - One of the best experts on this subject based on the ideXlab platform.
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Preliminary Study of an Immunochromatography Test for Serological Diagnosis of Canine Brucellosis
Reproduction in Domestic Animals, 2012Co-Authors: M M Wanke, Pablo C. Baldi, Fabián Cairó, Mariano Rossano, Mariano Laiño, N E Monachesi, E A Comercio, Marcela Martínez VivotAbstract:Contents The most widely used screening test for the diagnosis of Brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for Canine Brucellosis (FASTest® Brucella c., Megacor, Horbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute Brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic Brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic Brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID.
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Preliminary study of an immunochromatography test for serological diagnosis of Canine Brucellosis.
Reproduction in domestic animals = Zuchthygiene, 2012Co-Authors: M M Wanke, P C Baldi, Fabián Cairó, Mariano Rossano, Mariano Laiño, N E Monachesi, E A Comercio, Marcela Martínez VivotAbstract:The most widely used screening test for the diagnosis of Brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for Canine Brucellosis (FASTest(®) Brucella c., Megacor, Hörbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute Brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic Brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic Brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID.
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EVALUATION OF A NEW COMMERCIAL TEST FOR THE RAPID SEROLOGICAL DIAGNOSIS OF Canine Brucellosis
2011Co-Authors: M M Wanke, Pablo C. Baldi, Fabián Cairó, Mariano Rossano, Mariano Laiño, Marcela Martínez VivotAbstract:As in other species, the diagnosis of Brucellosis in the dog continues to be problematic. The most widely used screening test is the rapid slide agglutination test in the presence of 2mercaptoethanol (2ME-RSAT) using whole cells of the M(-) strain of Brucella canis as antigen. The diagnosis is partially confirmed by agar-gel immunodifusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases that may not be detected by these tests may be detected by ELISA tests that use a hot-saline extract (HS) or cytosolic proteins of Brucella as antigens (CP). The use of 2ME-RSAT in the routine clinical practice is complicated by the need of a microscope to read the reaction and an experienced operator to interpret the results. An immunochromatographic diagnostic test for Canine Brucellosis (FASTest® Brucella c., Megacor, Horbranz, Austria) has been recently released, which is very simple to perform and could be potentially used in the routine clinical practice. In the present study we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs.
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Use of enrofloxacin in the treatment of Canine Brucellosis in a dog kennel (clinical trial).
Theriogenology, 2006Co-Authors: M M Wanke, M V Delpino, P C BaldiAbstract:To date, no totally effective antibiotic for the eradication of Canine Brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0-2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating Canine Brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of Canine Brucellosis.
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Canine Brucellosis.
Animal reproduction science, 2004Co-Authors: M M WankeAbstract:This review discusses the prevalence, etiology, pathogenesis, clinical findings, diagnostic methods, therapy, management and public health considerations of Brucella canis infection in dogs. Canine Brucellosis is a contagious infection produced by a gram-negative coccobacilus called Brucella canis. The main sources of infection are vaginal fluids of infected females and urine in males. Routes of entry are venereal, oronasal, conjunctivae mucosa and placenta. The most significant symptoms are late abortions in bitches, epididymitis in males and infertility in both sexes, as well as generalized lymphadenitis, discospondylitis and uveitis. Diagnosis is complex because serology can give false positive results and chronic cases can give negative results, needing to be complemented with bacteriological studies. No antibiotic treatment is 100% effective and the infection often recurs in animals apparently treated successfully. Infected animals must be removed from the kennels and no longer used for breeding. Preferably, males should be castrated and females spayed. Human contagion is not frequent, although it has been reported, and is easily treated.
Pablo C. Baldi - One of the best experts on this subject based on the ideXlab platform.
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DISPATCHES Human Infection with M- Strain of
2013Co-Authors: Brucella Canis, Pablo C. Baldi, Jorge C. Wallach, Guillermo H, Carlos A. FossatiAbstract:The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of Canine Brucellosis. While this strain is avirulent in dogs, we report the case of clinical Brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production. Brucella canis is the causative agent of Canine Brucellosis, which causes contagious abortion, orchiepididymitis, and uveitis. Transmission to human requires close contact with infected animals or bacterial cultures. Symptomatic human infections are rare, probably because of the low virulence of B. canis; 31 human cases have been reported (1). In contrast to other Brucella species, which are pathogenic for humans (B. abortus, B. melitensis, B. suis) and yield smooth colonies, B. canis colonies are naturally rough. Therefore, serologic tests that use suspensions of smooth brucellae are not useful in diagnosing B. canis infections (2). Since suspensions of wild-type B. canis tend to aggregate even in the absence of specific antibodies, a less mucoid variant termed M-, which does not produce autoagglutination is used for serologic diagnosis (3). The M- strain has reduced virulence in dogs; even high doses of this strain do not induce the typical signs of Brucellosis in dogs (4). The pathogenic potential of the M- strain in humans remains unknown, and to the best of our knowledge, human infection by this strain has not been reported. We report a clinical and immunologic study of a human infection by the B. canis M- strain that shows that this strain can produce human disease similar to that produced by wild-type B. canis. Case Report A 35-year-old male laboratory worker was referred to a physician with recurrent fever, headache, arthralgia, weakness, and constipation, which had begun 1 month before. The patient worked in a laboratory that produced antigens for diagnostic use. Three weeks before symptoms began, he had been handling a dense culture of live B. canis M
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Preliminary Study of an Immunochromatography Test for Serological Diagnosis of Canine Brucellosis
Reproduction in Domestic Animals, 2012Co-Authors: M M Wanke, Pablo C. Baldi, Fabián Cairó, Mariano Rossano, Mariano Laiño, N E Monachesi, E A Comercio, Marcela Martínez VivotAbstract:Contents The most widely used screening test for the diagnosis of Brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for Canine Brucellosis (FASTest® Brucella c., Megacor, Horbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute Brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic Brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic Brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID.
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EVALUATION OF A NEW COMMERCIAL TEST FOR THE RAPID SEROLOGICAL DIAGNOSIS OF Canine Brucellosis
2011Co-Authors: M M Wanke, Pablo C. Baldi, Fabián Cairó, Mariano Rossano, Mariano Laiño, Marcela Martínez VivotAbstract:As in other species, the diagnosis of Brucellosis in the dog continues to be problematic. The most widely used screening test is the rapid slide agglutination test in the presence of 2mercaptoethanol (2ME-RSAT) using whole cells of the M(-) strain of Brucella canis as antigen. The diagnosis is partially confirmed by agar-gel immunodifusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases that may not be detected by these tests may be detected by ELISA tests that use a hot-saline extract (HS) or cytosolic proteins of Brucella as antigens (CP). The use of 2ME-RSAT in the routine clinical practice is complicated by the need of a microscope to read the reaction and an experienced operator to interpret the results. An immunochromatographic diagnostic test for Canine Brucellosis (FASTest® Brucella c., Megacor, Horbranz, Austria) has been recently released, which is very simple to perform and could be potentially used in the routine clinical practice. In the present study we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs.
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HUMAN INFECTION WITH M- STRAIN OF BRUCELLA CANIS
Emerging infectious diseases, 2004Co-Authors: Jorge C. Wallach, Pablo C. Baldi, Guillermo H. Giambartolomei, Carlos A. FossatiAbstract:The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of Canine Brucellosis. While this strain is avirulent in dogs, we report the case of clinical Brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production.
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Occurrence and Potential Diagnostic Applications of Serological Cross-Reactivities between Brucella and Other Alpha-Proteobacteria
Clinical and diagnostic laboratory immunology, 2004Co-Authors: M. Victoria Delpino, Carlos A. Fossati, Pablo C. BaldiAbstract:Agrobacterium, Sinorhizobium, and Ochrobactrum are genera closely related to Brucella but, in contrast to the latter, are not pathogenic for humans and animals. We studied by an indirect enzyme-linked immunosorbent assay (ELISA) the reactivities of Brucellosis sera against cytosolic (CYT) and membrane (MA) antigens from these nonpathogenic bacteria, and we evaluated the potential usefulness of these cross-reactions for the diagnosis of Brucellosis in humans, sheep, cows, and dogs. Canine infection by Brucella canis was detected with high specificity by CYT antigen-based ELISAs (96% for Agrobacterium, 96% for Sinorhizobium, and 91% for Ochrobactrum), while sensitivity was variable (58% for Agrobacterium, 88% for Sinorhizobium, and 84% for Ochrobactrum). In addition, it was possible to diagnose Canine disease shortly after exposure to the pathogen (15 days). Similar results for Canine Brucellosis were obtained with MA antigens. In contrast, normal sera from humans, sheep, and cattle reacted strongly with all the antigens (CYT and MA antigens from the three bacteria), producing high cutoff values and, consequently, low sensitivities. While for some host species the reactivity patterns of normal sera by Western blotting were similar to those produced with sera from infected individuals, the reactivity pattern of bovine sera against Sinorhizobium meliloti antigens exhibited some differential bands for the two groups of sera. These results show that crude fractions from nonpathogenic alpha-proteobacteria can be used to diagnose Canine Brucellosis but may need to be further separated into simpler fractions to have diagnostic usefulness in ovine, bovine, or human infection. By reducing the biosafety requirements, the use of antigens derived from these nonpathogenic bacteria would simplify the production of diagnostic kits for Brucellosis, especially in settings where biosafety level-3 facilities are scarce or absent.
Rodrigo Martins Soares - One of the best experts on this subject based on the ideXlab platform.
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MALDI-TOF MS and genomic analysis can make the difference in the clarification of Canine Brucellosis outbreaks
Scientific Reports, 2020Co-Authors: David Attuy Vey Da Silva, Lara Borges Keid, Rodrigo Martins Soares, Julia Teresa Ribeiro De Lima, Holger Brendebach, Josephine Grützke, Ralf Dieckmann, Dirk Hofreuter, Sascha Al DahoukAbstract:Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine Brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of Brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with Canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of Canine Brucellosis.
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Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for Canine Brucellosis diagnosis
Molecular and cellular probes, 2018Co-Authors: Maria Cryskely Agra Batinga, Rodrigo Martins Soares, Julia Teresa Ribeiro De Lima, Fábio Gregori, J. A. Diniz, Kerstin Muner, Trícia Maria Ferreira De Sousa Oliveira, Helena Ferreira, Lara Borges KeidAbstract:Canine Brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of Brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for Canine Brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of Canine Brucellosis.
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Comparison of three methods for recovery of Brucella canis DNA from Canine blood samples.
Journal of microbiological methods, 2017Co-Authors: Maria Cryskely Agra Batinga, Rodrigo Martins Soares, Julia Teresa Ribeiro De Lima, Kerstin Muner, Trícia Maria Ferreira De Sousa Oliveira, David Attuy Vey Da Silva, Jaíne C. Dos Santos, M. F. D Bigotto, Thalita Faita, Helena FerreiraAbstract:Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes Canine Brucellosis. Direct methods are the most appropriate for the detection of Canine Brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs.
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Evaluation of an Immunochromatographic Test to the Diagnosis of Canine Brucellosis Caused by Brucella canis.
Reproduction in domestic animals = Zuchthygiene, 2015Co-Authors: Lara Borges Keid, J. A. Diniz, Trícia Maria Ferreira De Sousa Oliveira, Helena Ferreira, Rodrigo Martins SoaresAbstract:This study evaluated the performance of an immunochromatographic test (ICT) for the diagnosis of Canine Brucellosis caused by Brucella canis, comparing its results with that of the rapid slide agglutination test with and without the use of 2-mercaptoethanol and the agar gel immunodiffusion test (AGID). The microbiological culture, PCR and clinical examination were used as reference. According to the results obtained in clinical examination, blood culture, culture of semen and vaginal swab and PCR in blood, semen and vaginal swab, a total of 102 dogs were divided into three groups: B. canis-infected dogs (Group 1), B. canis-non-infected dogs (Group 2) and dogs with suspected Brucellosis (Group 3). The diagnostic sensitivity of RSAT, 2ME-RSAT, AGID and ICT in Group 1 was, respectively, 75%, 37.5%, 27.8% and 89.58%. The diagnostic specificity of RSAT, 2ME-RSAT, AGID and ICT in Group 2 was, respectively, 91%, 100%, 100%, and 100%. In dogs with suspected Brucellosis, 9.67% were RSAT positive, none was positive by 2ME-RSAT, 3.22% were AGID positive and 6.45% were ICT positive. The main drawback concerning Canine Brucellosis diagnosis is the lack of a highly sensitive serological assay to be used as a screening test to the rapid identification of infected animals. The ICT showed a high diagnostic specificity and a diagnostic sensitivity value greater than that observed in the RSAT, 2ME-RSAT and AGID. However, 10.41% of infected dogs had negative results by ICT. These dogs were positive by microbiological culture and/or PCR, indicating active infection and consequently a higher potential of spreading Brucella. Although rapid and simple to perform, the ICT lacked sensitivity to be used as a screening test.
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Comparison of a PCR assay in whole blood and serum specimens for Canine Brucellosis diagnosis.
The Veterinary record, 2010Co-Authors: Lara Borges Keid, Rodrigo Martins Soares, Silvio Arruda Vasconcellos, Jane Megid, Vanessa Riesz Salgado, Leonardo Jose RichtzenhainAbstract:The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of Canine Brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected Brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected Brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of Canine Brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.
Leonardo Jose Richtzenhain - One of the best experts on this subject based on the ideXlab platform.
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Comparison of a PCR assay in whole blood and serum specimens for Canine Brucellosis diagnosis.
The Veterinary record, 2010Co-Authors: Lara Borges Keid, Rodrigo Martins Soares, Silvio Arruda Vasconcellos, Jane Megid, Vanessa Riesz Salgado, Leonardo Jose RichtzenhainAbstract:The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of Canine Brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected Brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected Brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of Canine Brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.
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comparison of agar gel immunodiffusion test rapid slide agglutination test microbiological culture and pcr for the diagnosis of Canine Brucellosis
Research in Veterinary Science, 2009Co-Authors: Lara Borges Keid, Rodrigo Martins Soares, Silvio Arruda Vasconcellos, Jane Megid, Vanessa Riesz Salgado, Leonardo Jose RichtzenhainAbstract:The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of Brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected Brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected Brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for Brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of Canine Brucellosis.
