Glanders Diagnosis in an Asymptomatic Mare from Brazil: Insights from Serology, Microbiological Culture, Mass Spectrometry, and Genome Sequencing.

This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders.

Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice.

Burkholderia mallei is an aerobic, Gram-negative, non-motile bacillus. As an obligate mammalian pathogen, it primarily affects solipeds. Although rarely transmitted to humans, the disease it causes, glanders, is classified as a zoonosis. The bacterium was officially eradicated in Brazil in 1969; however, it reemerged after three decades. This study aims to assess the virulence of a specific B. mallei strain, isolated in Brazil, in BALB/c mice through intranasal infection. The strain, B. mallei BAC 86/19, was obtained from the tracheal secretion of a young mare displaying positive serology but no clinical signs of glanders. Post-mortem examinations revealed macroscopic lesions consistent with the disease, however. In mice, the LD50 was determined to be approximately 1.59 × 105 colony-forming units (CFU)/animal. Mice exposed to either 0.1 × LD50 or 1 × LD50 displayed transient weight loss, which resolved after three or five days, respectively. B. mallei persisted within the liver and lung for five days post-infection and in the spleen for seven days. These findings underscore the detectable virulence of the Brazilian B. mallei BAC 86/19 strain in mice, which are relatively resilient hosts. This research points to the importance of the continued investigation of the virulence mechanisms and potential countermeasures associated with B. mallei infections, including their Brazilian isolates.

Molecular detection of Burkholderia mallei in different geographic regions of Brazil.

Glanders is a contagious disease of equids caused by the Gram-negative bacterium Burkholderia mallei. In Brazil, the disease is considered to be reemerging and has been expanding, with records of equids with positive serology in most of the federative units. However, there are few reports describing the genotypic detection of the agent. This study demonstrated the detection of B. mallei by species-specific PCR directly from tissues or from bacterial cultures, followed by amplicon sequencing in equids (equines, mules, and asinines) with positive serology for glanders in all five geographic regions of Brazil. The molecular evidence of B. mallei infection in serologically positive equids in this study expands the possibility of strain isolation and the conduction of epidemiological characterizations based on molecular information. The microbiological detection of B. mallei in cultures from nasal and palate swabs, even in equids without clinical manifestations, raises the possibility of environmental elimination of the agent.

Epidemiological Situation of Glanders in the State of Pará, Brazil.

Glanders is an anthropozoonosis caused by the bacteria Burkholderia mallei, affecting mainly equids. It has been eradicated in North America, Australia, and Western Europe, but continues to occur sporadically in countries in Asia, Africa, the Middle East, and South America. Its notification is mandatory by the World Organization for Animal Health. After 30 years, the disease reappeared in Brazil in 1999 and, thereafter, 1,413 outbreaks have been reported. However, the epidemiological situation of the disease in the country is not adequately known. Thus, 2718 animals from 654 properties in the state of Pará were randomly selected by sampling and examined using a serial protocol with Complement Fixation and Western Blot serological tests. The prevalence of properties infected with glanders in the state was estimated at 1.68% [0.84; 3.33] and of seropositive animals at 0.50% [0.27; 0.94]. The introduction of animals was individualized as a risk factor for disease introduction in the properties (OR = 5.9 [1.4; 25.5]). Despite the low prevalence of infected properties and seropositive animals, the state must review actions to fight the disease, considering that the strategies implemented have not affected the endemic balance of the disease. This process must involve all public and private agents interested in the topic.

Pathology, microbiology, and molecular evaluation of tissues from equids serologically positive for Burkholderia mallei in Midwestern Brazil / Avaliação patológica, microbiológica e molecular em amostras de tecidos de equídeos sorologicamente positivos para Burkholderia mallei no Centro-Oeste do Brasil

Glanders is a disease caused by the bacterium Burkholderia mallei that primarily affects horses, mules and donkeys. The disease can cause lesions in the skin, lungs and several other organs. However, it often manifests as an asymptomatic disease. In Brazil, serological tests of high sensitivity and specificity are used to assist in the detection of antibodies against B. mallei and to contribute to the control of the disease. However, due to the mandatory euthanasia of seroreactive animals, equids with positive serology for B. mallei and asymptomatic generated great conflicts between breeders, veterinarians and diagnostic laboratories. This study clarifies the limitations of complementary diagnostic tests for detecting B. mallei. It describes the clinical, morphological and laboratory findings in 24 equines from different municipalities in the Mato Grosso State, Brazil, which reacted to the complement fixation test and were positive in the western blotting test for glanders. Data and tissue samples were collected from 24 horses for histological, microbiological and molecular analysis. In 23 horses, no clinical signs, morphological alterations, microbiological isolation, or molecular detection would characterize B. mallei infection. On the other hand, samples from an asymptomatic horse without lesional alterations showed sequence amplification compatible with B. mallei in the PCR. Considering that the infection by B. mallei is subject to the application of animal sanitary defense measures and that, by international requirement and national legislation, the serological results are tools that should support the sanitation procedures for the error of the bacteria in the Mato Grosso State, Brazil.

Mormo é uma enfermidade causada pela bactéria Burkholderia mallei que acomete primariamente cavalos, mulas e burros. A doença pode causar lesões na pele, pulmões e em diversos outros órgãos, entretanto frequentemente manifesta-se como uma enfermidade assintomática. No Brasil são utilizados testes sorológicos de elevada sensibilidade e especificidade para auxiliar na detecção de anticorpos contra B. mallei e contribuir para controle da doença. Porém, devido à obrigatoriedade da eutanásia de animais sororeagentes, os equídeos com sorologia positiva para B. mallei e assintomáticos geraram grandes embates entre criadores, médicos-veterinários e laboratórios de diagnóstico. Este trabalho esclarece as limitações dos testes diagnósticos complementares para detecção de B. mallei e descreve os achados clínicos, morfológicos e de exames laboratoriais em 24 equídeos, procedentes de diferentes municípios do estado de Mato Grosso, Brasil, que reagiram ao teste de fixação de complemento e foram positivos no teste de "western blotting" para mormo. Foram colhidos dados e amostras de tecidos de 24 equídeos para análise histológica, microbiológica e molecular. Em 23 equídeos não existiam sinais clínicos, alterações morfológicas, isolamento microbiológico ou detecção molecular que caracterizassem infecção por B. mallei. Por outro lado, amostras de um cavalo assintomático e sem alterações lesionais apresentaram amplificação de sequência compatível com B. mallei na PCR. Considerando que a infecção por B. mallei é passível da aplicação de medidas de defesa sanitária animal e que por exigência internacional e da legislação nacional, os resultados sorológicos são ferramentas que devem amparar os procedimentos de saneamento para erradicação da bactéria no estado de Mato Grosso, Brasil.

Current status of glanders in Brazil: recent advances and challenges.

Glanders is an infectious disease that causes serious damage to the equine production chain in countries where it occurs endemically and poses a risk to public health. This study aimed to conduct an integrative review of the advances in the knowledge of glanders in Brazil over the last three decades since its re-emergence. Documentary research was conducted for the period between the years 2000 and 2022. SCOPUS and PUBMED databases were used to search for scientific articles, dissertations, and thesis, in addition to the Brazilian Digital Library of Thesis and Dissertations (BDTD). A total of 41 documents were retrieved, including 12 dissertations, five theses, and 24 scientific articles. The Federal Rural University of Pernambuco (UFRPE) group provided a noteworthy amount of material on this topic (25 documents, four theses, three dissertations, and 18 scientific articles). During this period, ten dissertations, one thesis, and six scientific articles published by other groups in other states of the Federation were also identified. It was concluded that there was a significant number of scientific publications with relevant data on the clinical, epidemiological, pathological, microbiological, serological, and molecular characteristics of glanders disease in Brazil. Additionally, training of human resources regarding this disease led to an increase in the nucleation of research groups, especially in the northeast region of Brazil. Despite significant advances, new research groups and specific funding are still needed for the development of more accurate diagnostic methods, immunizing production, training of veterinarians to recognize the disease, and more robust programs to control and eradicate the disease in Brazil.

Glanders and brucellosis in equids from the Amazon region, Brazil.

Glanders and brucellosis are zoonotic infectious diseases that affect equids in several countries worldwide. On Marajó Island (Amazon region of Brazil), Marajoara and Puruca horses, which are well adapted to the climatic and territorial adversities of the region, play a fundamental role in the local economy and in the sociocultural lives of the population. However, these animals have undergone a drastic reduction in number, markedly due to precarious veterinary care, unknown causes of morbidity and mortality, and disordered crossing with other breeds introduced to the island. Thus, this study aimed to investigate the occurrence of glanders and brucellosis in equids on a property located in the municipality of Soure, Marajó Island (Brazil). Serum samples were collected from 388 animals (357 horses and 31 mules), maintained in an extensive breeding system, in a property that was also extensively breeding buffaloes, goats, and sheep, with contact among species. The sera were tested for glanders using an indirect ELISA (ELISAi), and the results were confirmed by immunoblotting. The diagnosis of brucellosis was made using the Rose Bengal test (RBT) and confirmed through the Serum Agglutination test (SAT) and 2-mercaptoethanol test. In the case of glanders, 2.31% (9/388) of animals were positive in ELISAi test, of which eight had results confirmed by immunoblotting, representing 2.06% seropositivity in the entire herd. For brucellosis, serum samples from 6.7% (26/388) horses were reactive in the RBT, of which 4.12% (18/388) had a titer ≥50 and 2.06% (8/388) had a titer ≥100 in the SAT. This is the first study to report the occurrence of glanders and equine brucellosis in the municipality of Soure/Marajó Island. Monitoring the occurrence of such diseases is extremely important since they affect the herds economically and zootechnically, in addition to their high zoonotic potential. The number of animals sampled in this study, as well as the way they are raised and managed, is representative of the total equid population of the island. These results, combined with previous studies on buffaloes, indicate that these diseases are endemic in the Marajo Island.

Situational profile of equidae breeders in the municipality of Soure, Pará: an approach to glanders and adoption of relevant sanitary measures / Perfil situacional dos criadores de equídeos do município de Soure, Pará: uma abordagem quanto ao mormo e a adoção das medidas sanitárias pertinentes

A survey was conducted on the knowledge, attitudes, and behaviors of Equidae breeders in the municipality of Soure, Marajó Island, Pará, regarding animal health problems in the properties, especially glanders and adoption of sanitary measures that are pertinent to their combat, to elaborate on educational technical material with the appropriate methodology. The study included 50 interviewees from urban and rural areas of this municipality. Regarding data collection, structured interviews containing 22 multiple choice questions on the socioeconomic characteristics of breeders and/or owners of Equidae, breeding habits and models, and conceptions about sanitary control and their relationship with the animal health defense agency were conducted. From the qualitative analysis of data, 66% (33/50) interviewees were identified as literate, which facilitates the choice of the best language to use for communication. Regarding the choice of the best language for the transfer of information, 62% (31/50) interviewees said that a mobile phone is currently the best tool for transmitting information. Further, 40% (20/50) interviewees breed their Equidae extensively, raising an alert for epidemiological surveillance, and only 10% (5/50) interviewees turned to ADEPARÁ when the animals were sick. Based on the study results, Equidae breeders need guidance regarding the actions of the animal health defense services, and sanitary education is a fundamental measure for raising awareness and clarification of glanders to mitigate damage to public health and animal welfare, avoiding its spread in the State.(AU)

Foi realizado um levantamento sobre conhecimentos, atitudes e comportamentos dos criadores de equídeos do município de Soure, na Ilha do Marajó, Pará, quanto aos problemas sanitários nas propriedades, em especial, ao mormo e a adoção de medidas sanitárias pertinentes ao seu combate - elaboração de material técnico educativo com metodologia ade-quada ao público-alvo trabalhado. A pesquisa contou com 50 entrevistados pertencentes às zonas urbana e rural desse muni-cípio. Quanto a coleta de dados, foram realizadas entrevistas estruturadas contendo 22 questões de múltipla escolha referentes às características socioeconômicas dos criadores e/ou proprietários de equídeos; aos hábitos e aos modos de criação; às con-cepções sobre o controle sanitário e a relação destes com o órgão de defesa sanitária animal. Da análise qualitativa dos dados, observou-se que 66% (33/50) dos entrevistados são alfabetizados; o que facilita a escolha da melhor linguagem para o repasse de informações; 62% (31/50) elencou que o celular, atualmente, é a melhor ferramenta para a transmissão de informações; 40% (20/50) cria seus equídeos de maneira extensiva, representando um alerta à vigilância epidemiológica e apenas 10% (5/50) recorre à ADEPARÁ quando os animais adoecem. Com base no estudo, foi possível concluir que os criadores de equídeos necessitam de orientações quanto às ações do serviço de defesa sanitária animal, sendo a educação sanitária medida fundamen-tal para conscientização e esclarecimento deles sobre o mormo, de maneira a mitigar seus danos à saúde pública e ao bem-estar dos animais, evitando sua propagação no Estado.(AU)

Validation of three qPCR for the detection of Burkholderia mallei in equine tissue samples.

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.

Clinical repercussions of Glanders (Burkholderia mallei infection) in a Brazilian child: a case report

Abstract Glanders is a relatively unknown zoonotic disease caused by Burkholderia mallei. This bacterium affect solipeds and humans, and can be used as a biological warfare. Glanders is characterized as an occupational disease. We report the case of an 11-year-old boy who was presented to an emergency department with chest pain and dyspnea. He evolved into septic shock, pneumonia, and multiple abscesses. B. mallei was found in the exudate culture. Human infection is rare and difficult to confirm. The knowledge on glanders is important for differential diagnosis from other serious illnesses causing pneumonia and multiple abscesses.

First record of Burkholderia mallei Turkey 10 strain originating from glanderous horses from Brazil.

Burkholderia (B.) mallei is the causative agent of glanders in Equidae. This study describes the first record of the Turkey 10 strain of B. mallei in glanderous horses in Northeastern of Brazil. This description should contribute to the future actions of diagnosis, control, and eradication of this disease in Brazil.

Spatiotemporal Analysis of Glanders in Brazil.

In Brazil, glanders remains a serious problem, with the obligatory sacrifice of disease-positive animals without compensation. Each year, glanders cases are reported in several regions of the country, causing severe economic losses and trade restrictions. The present study describes and discusses the occurrence of glanders foci in Brazil during a 12-year period from 2005 to 2016. The highest frequency of reported affected holdings during the study period was in the northeast region. Moreover, during this period, the disease incidence in Brazil showed an overall increasing tendency. The number of affected holdings significantly increased during the last four years of the period, and more cases were noted during the months of May and June. Spatiotemporally, there are four high-risk glanders clusters: (1) cluster A (relative risk [RR = 6.51, P < .0001) involved the northeast region from March 2008 to February 2014; (2) cluster B (RR = 17.37, P < .0001) involved a southeast region state from March 2013 to June 2015; (3) cluster C (RR = 6.92, P < .0001) involved the states in the midwest, southeast, and south regions of Brazil from March 2015 to May 2016; and (4) cluster D (RR = 19.07, P < .0001) involved a north region state from October 2015 to April 2016. Only two states of the north region (Acre and Amapá) did not experience glanders during the study period.

Burkholderia mallei: The dynamics of networks and disease transmission.

Glanders is a highly infectious zoonotic disease caused by Burkholderia mallei. The transmission of B. mallei occurs mainly by direct contact, and horses are the natural reservoir. Therefore, the identification of infection sources within horse populations and animal movements is critical to enhance disease control. Here, we analysed the dynamics of horse movements from 2014 to 2016 using network analysis in order to understand the flow of animals in two hierarchical levels, municipalities and farms. The municipality-level network was used to investigate both community clustering and the balance between the municipality's trades and the farm-level network associations between B. mallei outbreaks and the network centrality measurements, analysed by spatio-temporal generalized additive model (GAM). Causal paths were established for the dispersion of B. mallei outbreaks through the network. Our approach captured and established a direct relationship between movement of infected equines and predicted B. mallei outbreaks. The GAM model revealed that the parameters in degree and closeness centrality out were positively associated with B. mallei. In addition, we also detected 10 communities with high commerce among municipalities. The role of each municipality within the network was detailed, and significant changes in the structures of the network were detected over the course of 3 years. The results suggested the necessity to focus on structural changes of the networks over time to better control glanders disease. The identification of farms with a putative risk of B. mallei infection using the horse movement network provided a direct opportunity for disease control through active surveillance, thus minimizing economic losses and risks for human cases of B. mallei.

Sinais clínicos em equídeos naturalmente infectados por Burkholderia mallei no estado de Alagoas: relato de caso / Clinical Signs in equids naturally infected by Burkholderia mallei in the state of Alagoas: case report / Signos Clínicos en équidos infectados naturalmente por Burkholderia mallei en el estado de Alagoas: reporte de caso

Há alguns anos o mormo chegou a ser considerado erradicado no Brasil pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA), porém tem ganhado importância devido ao aumento no número de casos nos últimos anos em todos os estados brasileiros. Os sinais clínicos foram reconhecidos desde que a doença foi registrada pela primeira vez por Hipócrates e Aristóteles e as formas de manifestação da doença são prepatente, hiperaguda, aguda e crônica. Objetivou-se relatar os achados clínicos de equídeos naturalmente infectados com mormo em propriedades consideradas foco pela Agência de Defesa e Inspeção Agropecuária de Alagoas - ADEAL e pelo MAPA no estado de Alagoas. Os sinais clínicos observados nos animais acometidos foram, apatia, dispneia estertorosa, nódulos linfáticos infartados (aumentados), expectoração nasal mucopurulenta bilateral em 3/16 dos animais observados, 1/16 animal apresentou a forma crônica evidenciada pelo edema de membro posterior esquerdo, com presença de lesões ulceradas, linfagite disseminada, expectoração de secreção nasal serosanguinolenta bilateral (hemoptise) e dispneia severa. Por fim os sinais clínicos e achados macroscópicos observados nos animais naturalmente infectados por Burkholderia mal/ei no estado de Alagoas corroboram com o descrito na literatura veterinária e para que haja o controle e a não disseminação da doença, animais positivos devem ser eutanasiados e propriedades foco devem ser interditadas.(AU)

A few years ago the glanders came to be considered eradicated in Brazil by the Ministry of Agriculture, Livestock and Food Supply (MAPA), but has gained importance due to the increase in the number of cases in recent years in ali Brazilian states. The clinical signs were recognized since the disease was first recorded by Hippocrates and Aristotle and the forms of the disease manifestation are prepatente, hyperacute, acute and chronic. This study aimed to report the clinical findings of equids naturally infected with glanders properties considered focus by the Defense Agency and Agriculturallnspection of Alagoas - ADEAL and the ap in the state of Alagoas. The clinical signs observed in animais affected were, apathy, dyspnea stertorous, infarcted Iymph nodes (increased), expectoration mucopurulent bilateral nasal in 3/16 of animais observed, 1/16 animal presented the chronic form evidenced by edema in the left posterior limb, with the presence of ulcerated lesions, linfagite disseminated, the expectoration of nasal secretion serosanguinolenta bilateral (hemoptysis) and severe dyspnea. Finally, the clinical signs and macroscopic findings observed in animais naturally infected by Burkholderia mallei in Alagoas state corroborate with the previously described in the literature on veterinary and for which there is no control and the spread of the disease, positive animais should be euthanized and focus should be interditadas properties.(AU)

Hace unos anos el Muermo llegó a ser considerado erradicado en Brasil por el Ministerio de Agricultura, Ganadería y Alimentación (MAPA), pero ha adquirido mayor importancia debido al aumento en el número de casos en los últimos anos en todos los estados brasilenos. Los signos clínicos fueron reconocidos desde que la enfermedad fue grabado por primera vez por Hipócrates y Aristóteles y las formas de las manifestaciones de la enfermedad son prepatente, Hiperaguda, aguda y crónica. Este estudio tiene como objetivo informar los hallazgos clínicos de équidos infectados naturalmente con el Muermo consideran propiedades focus por Ia Agencia de Defensa e Inspección Agrícola de Alagoas - ADEAL Y el MAPA en el estado de Alagoas. Los signos clínicos observados en los animales afectados fueron, apatía, disnea estertorosa, ganglios linfáticos infartados (aumento), expectoración nasal mucopurulento bilateral en 3/16 (tres/dieciséis) de animales observados, 1/16 (uno/dieciséis) animal presentado Iaforma crónica evidenciada por edema en la extremidad posterior izquierda, con la presencia de lesiones ulceradas, linfagite difundido, la expectoración de secreción nasal serosanguinolenta bilateral (hemoptisis) y disnea severa. Por último, los signos clínicos y los hallazgos macroscópicos observados en los animales infectados naturalmente por Burkholderia mallei en el Estado de Alagoas corroborar con las descritas previamente en la literatura sobre veterinaria y para los cuales no hay control y la propagación de la enfermedad, los animales positivos deben ser sacrificados y el enfoque debe ser interditadas propiedades.(AU)

Sinais clínicos em equídeos naturalmente infectados por Burkholderia mallei no estado de Alagoas: relato de caso / Clinical Signs in equids naturally infected by Burkholderia mallei in the state of Alagoas: case report / Signos Clínicos en équidos infectados naturalmente por Burkholderia mallei en el estado de Alagoas: reporte de caso

Há alguns anos o mormo chegou a ser considerado erradicado no Brasil pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA), porém tem ganhado importância devido ao aumento no número de casos nos últimos anos em todos os estados brasileiros. Os sinais clínicos foram reconhecidos desde que a doença foi registrada pela primeira vez por Hipócrates e Aristóteles e as formas de manifestação da doença são prepatente, hiperaguda, aguda e crônica. Objetivou-se relatar os achados clínicos de equídeos naturalmente infectados com mormo em propriedades consideradas foco pela Agência de Defesa e Inspeção Agropecuária de Alagoas - ADEAL e pelo MAPA no estado de Alagoas. Os sinais clínicos observados nos animais acometidos foram, apatia, dispneia estertorosa, nódulos linfáticos infartados (aumentados), expectoração nasal mucopurulenta bilateral em 3/16 dos animais observados, 1/16 animal apresentou a forma crônica evidenciada pelo edema de membro posterior esquerdo, com presença de lesões ulceradas, linfagite disseminada, expectoração de secreção nasal serosanguinolenta bilateral (hemoptise) e dispneia severa. Por fim os sinais clínicos e achados macroscópicos observados nos animais naturalmente infectados por Burkholderia mal/ei no estado de Alagoas corroboram com o descrito na literatura veterinária e para que haja o controle e a não disseminação da doença, animais positivos devem ser eutanasiados e propriedades foco devem ser interditadas.

A few years ago the glanders came to be considered eradicated in Brazil by the Ministry of Agriculture, Livestock and Food Supply (MAPA), but has gained importance due to the increase in the number of cases in recent years in ali Brazilian states. The clinical signs were recognized since the disease was first recorded by Hippocrates and Aristotle and the forms of the disease manifestation are prepatente, hyperacute, acute and chronic. This study aimed to report the clinical findings of equids naturally infected with glanders properties considered focus by the Defense Agency and Agriculturallnspection of Alagoas - ADEAL and the ap in the state of Alagoas. The clinical signs observed in animais affected were, apathy, dyspnea stertorous, infarcted Iymph nodes (increased), expectoration mucopurulent bilateral nasal in 3/16 of animais observed, 1/16 animal presented the chronic form evidenced by edema in the left posterior limb, with the presence of ulcerated lesions, linfagite disseminated, the expectoration of nasal secretion serosanguinolenta bilateral (hemoptysis) and severe dyspnea. Finally, the clinical signs and macroscopic findings observed in animais naturally infected by Burkholderia mallei in Alagoas state corroborate with the previously described in the literature on veterinary and for which there is no control and the spread of the disease, positive animais should be euthanized and focus should be interditadas properties.

Hace unos anos el Muermo llegó a ser considerado erradicado en Brasil por el Ministerio de Agricultura, Ganadería y Alimentación (MAPA), pero ha adquirido mayor importancia debido al aumento en el número de casos en los últimos anos en todos los estados brasilenos. Los signos clínicos fueron reconocidos desde que la enfermedad fue grabado por primera vez por Hipócrates y Aristóteles y las formas de las manifestaciones de la enfermedad son prepatente, Hiperaguda, aguda y crónica. Este estudio tiene como objetivo informar los hallazgos clínicos de équidos infectados naturalmente con el Muermo consideran propiedades focus por Ia Agencia de Defensa e Inspección Agrícola de Alagoas - ADEAL Y el MAPA en el estado de Alagoas. Los signos clínicos observados en los animales afectados fueron, apatía, disnea estertorosa, ganglios linfáticos infartados (aumento), expectoración nasal mucopurulento bilateral en 3/16 (tres/dieciséis) de animales observados, 1/16 (uno/dieciséis) animal presentado Iaforma crónica evidenciada por edema en la extremidad posterior izquierda, con la presencia de lesiones ulceradas, linfagite difundido, la expectoración de secreción nasal serosanguinolenta bilateral (hemoptisis) y disnea severa. Por último, los signos clínicos y los hallazgos macroscópicos observados en los animales infectados naturalmente por Burkholderia mallei en el Estado de Alagoas corroborar con las descritas previamente en la literatura sobre veterinaria y para los cuales no hay control y la propagación de la enfermedad, los animales positivos deben ser sacrificados y el enfoque debe ser interditadas propiedades.

First molecular characterisation of a Brazilian Burkholderia mallei strain isolated from a mule in 2016.

We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.

Produção e purificação parcial de PPD-maleína para diagnóstico do mormo em equídeos / Partially purified malleo-proteins production for glanders diagnosis in Equidae

Objetivou-se com este estudo produzir e purificar parcialmente a PPD-maleína a partir de amostras de Burkholderia mallei isoladas de equídeos no Brasil com potencial para uso no diagnóstico do mormo. As linhagens de B. mallei fenotipicamente caracterizadas e de virulência comprovada foram inoculadas em caldo Dorset-Henley para crescer e metabolizar. Em seguida, as proteínas foram separadas por precipitação com ácido tricloroacético e precipitadas com sulfato de amônia. As PPDs-maleínas foram concentradas em 1,0mg/mL e na avaliação realizada em cobaios foi eficaz no desenvolvimento da hipersensibilidade do tipo tardia e consequentemente na identificação de animais verdadeiro positivos e exclusão dos verdadeiro negativos, sendo uma possibilidade em potencial para utilização no diagnóstico do mormo.(AU)

The objective of this study was to produce and partially purify Malleo-protein from Burkholderia mallei samples isolates from Equidae in Brazil with potential for use in the diagnosis of glanders. The strain B. mallei phenotypically characterized and proven virulent was inoculated into broth Dorset-Henley to grow and metabolize. The proteins were separated by trichloroacetic acid precipitation and amonium sulfate precipitation. The PPD mallein was concentrated 1.0mg/mL and biologically tested in guinea pigs. It was effective in the development of delayed-type hypersensitivity and consequently to identify true-positive animals and to exclude of true negatives. There is the possibility for potential use in the glanders diagnosis in Equidae.(AU)

Produção e purificação parcial de PPD-maleína para diagnóstico do mormo em equídeos / Partially purified malleo-proteins production for glanders diagnosis in Equidae

Objetivou-se com este estudo produzir e purificar parcialmente a PPD-maleína a partir de amostras de Burkholderia mallei isoladas de equídeos no Brasil com potencial para uso no diagnóstico do mormo. As linhagens de B. mallei fenotipicamente caracterizadas e de virulência comprovada foram inoculadas em caldo Dorset-Henley para crescer e metabolizar. Em seguida, as proteínas foram separadas por precipitação com ácido tricloroacético e precipitadas com sulfato de amônia. As PPDs-maleínas foram concentradas em 1,0mg/mL e na avaliação realizada em cobaios foi eficaz no desenvolvimento da hipersensibilidade do tipo tardia e consequentemente na identificação de animais verdadeiro positivos e exclusão dos verdadeiro negativos, sendo uma possibilidade em potencial para utilização no diagnóstico do mormo.

The objective of this study was to produce and partially purify Malleo-protein from Burkholderia mallei samples isolates from Equidae in Brazil with potential for use in the diagnosis of glanders. The strain B. mallei phenotypically characterized and proven virulent was inoculated into broth Dorset-Henley to grow and metabolize. The proteins were separated by trichloroacetic acid precipitation and amonium sulfate precipitation. The PPD mallein was concentrated 1.0mg/mL and biologically tested in guinea pigs. It was effective in the development of delayed-type hypersensitivity and consequently to identify true-positive animals and to exclude of true negatives. There is the possibility for potential use in the glanders diagnosis in Equidae.

Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos / Standardization and evaluation of an indirect ELISA for the serological diagnosis of glanders in horses

O mormo é uma enfermidade infecto-contagiosa de caráter agudo ou crônico que acomete principalmente os equídeos, causando enormes prejuízos na cadeia produtiva do cavalo. Para controlar a enfermidade o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico oficial pela fixação do complemento (FC), maleinização e sacrifício dos animais positivos. Os kits atuais utilizados no diagnóstico da doença são importados, dificultando e encarecendo sua aplicação na rotina. Objetivou-se com este estudo padronizar um teste de ELISA indireto utilizando o extrato protéico de Burkholderia mallei isolada a partir de equídeo portador no estado de Pernambuco. As amostras foram cultivadas em ágar sangue 10%, incubada por 48h a 37°C; posteriormente caracterizou-se fenotípica e genotipicamente uma das colônias isoladas, e em seguida a cultivou em BHI para enriquecimento; logo após, esta foi repicada para o meio Dor-set Henley o qual foi incubado a 37ºC sob 60rpm por oito semanas. Para padronização do teste utilizou-se o Conjugado Proteína G Peroxidase Sigma na diluição de 1:90.000, com soros diluídos em 1:100 e o antígeno em 1:400. Utilizou-se 60 soros como controle negativo testados frente à FC para determinação do ponto de corte o qual ficou em 0,042nm. Feitas as padronizações, foram testadas 300 amostras, onde 99% (297) foram concordantes com os resultados obtidos na FC. Ao final, o ensaio apresentou 100% de sensibilidade e 98,2% de especificidade com valores preditivo positivo e negativo de 97,7% e 100%, respectivamente. O teste de concordância kappa foi 0,98 e a repetibilidade intra e interplaca ficaram em 8,8 e 10,3%, respectivamente. Diante dos resultados obtidos durante os ensaios, conclui-se que o teste de ELISA indireto pode ser utilizado como uma ferramenta de diagnóstico eficiente. Entretanto, mais ensaios devem ser realizados visando consolidar a confiabilidade do referido teste.(AU)

Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37°C; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.(AU)

Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos / Standardization and evaluation of an indirect ELISA for the serological diagnosis of glanders in horses

O mormo é uma enfermidade infecto-contagiosa de caráter agudo ou crônico que acomete principalmente os equídeos, causando enormes prejuízos na cadeia produtiva do cavalo. Para controlar a enfermidade o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico oficial pela fixação do complemento (FC), maleinização e sacrifício dos animais positivos. Os kits atuais utilizados no diagnóstico da doença são importados, dificultando e encarecendo sua aplicação na rotina. Objetivou-se com este estudo padronizar um teste de ELISA indireto utilizando o extrato protéico de Burkholderia mallei isolada a partir de equídeo portador no estado de Pernambuco. As amostras foram cultivadas em ágar sangue 10%, incubada por 48h a 37°C; posteriormente caracterizou-se fenotípica e genotipicamente uma das colônias isoladas, e em seguida a cultivou em BHI para enriquecimento; logo após, esta foi repicada para o meio Dor-set Henley o qual foi incubado a 37ºC sob 60rpm por oito semanas. Para padronização do teste utilizou-se o Conjugado Proteína G Peroxidase Sigma na diluição de 1:90.000, com soros diluídos em 1:100 e o antígeno em 1:400. Utilizou-se 60 soros como controle negativo testados frente à FC para determinação do ponto de corte o qual ficou em 0,042nm. Feitas as padronizações, foram testadas 300 amostras, onde 99% (297) foram concordantes com os resultados obtidos na FC. Ao final, o ensaio apresentou 100% de sensibilidade e 98,2% de especificidade com valores preditivo positivo e negativo de 97,7% e 100%, respectivamente. O teste de concordância kappa foi 0,98 e a repetibilidade intra e interplaca ficaram em 8,8 e 10,3%, respectivamente. Diante dos resultados obtidos durante os ensaios, conclui-se que o teste de ELISA indireto pode ser utilizado como uma ferramenta de diagnóstico eficiente. Entretanto, mais ensaios devem ser realizados visando consolidar a confiabilidade do referido teste.

Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37°C; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.