First serological diagnosis of Mycobacterium avium subsp. paratuberculosis infection in sheep in the state of Pernambuco, Brazil.

This study aimed to diagnose Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep in the state of Pernambuco, Brazil. A total of 276 blood samples were analyzed using the enzyme-linked immunosorbent assay IDEXX Paratuberculosis Screening kit, and 261 fecal samples were submitted for bacterial culture and polymerase chain reaction tests. An animal-level sero-frequency of 0.72% (n = 2/276) and a farm-level sero-frequency of 20% (n = 2/10) were found. All fecal sample cultures were negative, and molecular analyses were also negative. To the best of our knowledge, this is the first study of MAP infection in sheep in the state of Pernambuco and one of the pioneers in the country. It is an asymptomatic disease that is difficult to diagnose in this species because the susceptibility of sheep to the organism is lower than that of other ruminant species. However, the sero-frequency found reveals that there is MAP exposure in sheep flocks in the region. In addition, serological monitoring can contribute to the observation of the organism's behavior in herds. Our results support the potential risk of MAP infection in sheep in the state of Pernambuco, Brazil.

Status of paratuberculosis in beef cattle In Rio Grande do Sul, Brazil / [Status da paratuberculose em bovinos de corte no Rio Grande do Sul, Brasil]

RESUMO A paratuberculose em bovinos de corte já foi diagnosticada no estado do Rio Grande do Sul, mas relatada em apenas um rebanho. Nesse contexto, o objetivo desta pesquisa é verificar se a paratuberculose pode ser detectada em rebanhos de bovinos de corte de diferentes mesorregiões do RS, por meio do teste ELISA. As amostras foram coletadas de bovinos provenientes de seis mesorregiões, entre elas as maiores produtoras de bovinos de corte do RS. Apenas duas amostras foram classificadas como suspeitas no teste de ELISA, as outras 391 amostras foram negativas. Os resultados demonstram baixa detecção da doença nas mesorregiões testadas, com apenas 0,5% das amostras com resultado suspeito. Assim, esses dados demonstram que a enfermidade é pouco frequente nos rebanhos testados e que um programa de controle pode ser implementado de forma viável e economicamente lucrativa.

Presence of antibodies against Mycobacterium avium subspecies paratuberculosis in Brazilian high-producing dairy herds.

This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil.

Paratuberculosis control strategies in dairy cattle: A systematic review.

Background: Mycobacterium avium subsp. paratuberculosis is the causative agent of paratuberculosis (PTB), incurable enterocolitis, affecting domestic and wild ruminants. Economic losses, impacts on animal health and welfare, and public health concerns justify its herd-level control. Aim: To systematically collect information to answer: What are the control and eradication strategies of PTB in dairy cattle worldwide? Methods: The search procedure was carried out on October 2nd, 2019, and updated on August 3rd, 2021, using OVID®, SciELO, and Redalyc databases, and the registers from the International Colloquium on Paratuberculosis (1991-2018). The inclusion criteria considered articles published in English, Portuguese, and Spanish and in peer-reviewed journals. The exclusion criteria included irrelevant topics, species other-than bovines, and not original articles. Definitive studies were obtained through the consensus of the authors on eligibility and quality. Data extraction was performed, considering bibliographic information, control and outcome strategies, follow-up time, and results. Results: Twenty-six relevant studies were found, reporting the use of three grouped control strategies: hygiene and management strategy (HMS), test-and-cull strategy (TCS), and vaccination strategy (VS). The HMS was the most common one (20/26), followed by TCS (17/26) and VS (7/26). Combined control strategies such as TCS-HMS (12/26), TCS-VS (1/26), and HMS-VS (1/26) were also described, and the consideration of the three control strategies (TCS-HMS-VS) was reported in two articles. The HMS included practices such as neonates/juvenile livestock hygiene, biosecurity, prevention of infection introduction into the herd, and environmental management. Within HMS, the most frequent practices were to remove calves from their dams as soon as possible after birth and to keep the minimal exposure of calves and heifers to adult cattle. As limitations, within the HMS, it is considered that some strategies cannot be included due to lack of compliance, or the application of the same strategy among one study and another may have a different degree of interpretation; publication bias was not controlled since the results of the control programs in endemic countries may be not available. Conclusion: The main PTB control strategies in dairy cattle worldwide are HMS, TCS, and VS. The use of one or several combined strategies has been found to succeed in controlling the disease at the herd-level.

Mycobacterium avium subsp. paratuberculosis (MAP) in goat milk in the semiarid region of the Brazilian Northeast - molecular characterization

Background: Goat farming has been on the rise in Brazil in recent years. Overall, 93% of the national herd is concentrated in the Northeast, with the state of Paraíba being the largest goat milk producer in the country. Considering Mycobacterium avium subsp. paratuberculosis (MAP) as a sanitary issue for the development of animal farming with risks for human health and that is a notifiable disease, this research was structured with the objective of confirming the presence and performing a molecular characterization of MAP in goat milk destined for processing plants in the semiarid region of the Brazilian Northeast. Materials, Methods & Results: Samples from 179 production units and 5 collective bulk tanks and 4 samples of pasteurized goat milk were analyzed through Real-time polymerase chain reaction (qPCR). Genetic material (DNA) for MAP was found in the goat milk sample from 1 production unit (1/179). From this positive sample, 9 lactating goats were identified in the original property, 7 of which showed MAP DNA in milk samples (77.77%). The characterization of the nucleotide sequence detected in the positive sample has 99% identity with KJ173784. Discussion: One sample (1/179), from the production units, had MAP genetic material (DNA) detected using the molecular test. Samples from these production units represent the milk from all lactating goats from each producer. Therefore, it was possible to identify from which farm the samples originated, allowing individual animals to then be tested, with milk samples collected from 9 goats and MAP DNA detected in 7 of them (77.77%) via PCR. Control and/or prevention programs need this type of surveillance in reason that it allows the tracking of possible foci from milk samples collected from dairy products or cooling stations. The use of PCR to detect MAP foci via goat milk is thus advantageous because samples are obtained in a non-invasive manner, with faster results when compared to the culture technique. The low detection via PCR in goat milk may be related to factors such as the small amount of MAP eliminated and the intermittent excretion in asymptomatic animals, as also false-positive samples. Samples from the collective bulk tanks was negative. It is possible that the combination of milk from all the properties diluted the amount of MAP. This suggests that the sensitivity of the PCR can be improved if the samples are obtained from the pooled milk from the same property. In some regions of Brazil, for example, showed the frequency of Zona da Mata region of the state of Minas Gerais, Brazil, found 1.94% of positive samples (9/464) and 9.76% (4/41) of properties with at least 1 positive sample for MAP. Different results to what were found in the semiarid region of Paraíba, where climate and production characteristics are different. Goats are susceptible to 3 strains: type "S" (Sheep), "Bison type" and type "C" (Cattle). Previous contact with this species may explain the similarity between the strain found in goat milk and those detected from bovine samples. This must also be taken into consideration during diagnosis and upon implementation of control measures for paratuberculosis in goats. Mycobacterium avium subsp. paratuberculosis was recorded for the first time in goat milk in the semiarid region, which may reveal a potential biological risk to humans and suggests the need for active surveillance of the agent.

Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in cattle, sheep, and goats from Latin America and the Caribbean: a systematic review.

This study aimed to systematically collect and appraise the scientific evidence to answer the research question: What MAP genotypes have been isolated from cattle, sheep, and goats in Latin America and the Caribbean? An electronic search was conducted on three platforms (i.e., OVID®, Web of Science®, SciELO) as well as on the proceedings of the International Colloquium on Paratuberculosis. Inclusion and exclusion criteria were defined a priori and conserved through the systematic process and only articles published in peer-reviewed journals were considered. A total of 26 articles met the definitive inclusion criteria. All were published in English, in 15 different journals, and between 1989 and 2020. The relevant articles reported the use of six different genotyping techniques (i.e., polymerase chain reaction-restriction endonuclease analysis, restriction fragment length polymorphism, type-specific-PCR, mycobacterial interspersed repetitive units-variable number of tandem repeats, multi-locus short sequence repeat, single nucleotide polymorphism) in isolates from seven countries. Genotypes found so far in the region using typing techniques were mainly C type. MIRU-VNTR mostly reported INMV 1, INMV 2, and INMV 11 subtypes, among others. MLSSR reported genotypes from four different countries, reporting nine different subtypes of which 7g-10g-4ggt was the most common for loci 1, 2, and 8, respectively. Regardless the high diversity of techniques used so far to genotype Latin American and Caribbean MAP isolates, the original question of this systematic review has been answered. In addition, a relative genetic similarity between MAP strains recovered from cattle, goats, and sheep unrelatedly of the matrix and geographic origin was identified.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Tipificación molecular de Mycobacterium avium subsp. paratuberculosis en lecherías de Antioquia, Colombia

RESUMEN Objetivo. Determinar la diversidad molecular de Mycobacterium avium subsp. paratuberculosis (MAP) en muestras ambientales de hatos lecheros colombianos. Materiales y métodos. Las muestras ambientales de 25 hatos lecheros positivos a MAP por IS900-qPCR se cultivaron por duplicado en medio de yema de huevo de Herrold con micobactina J para obtener aislamientos. Las colonias sospechosas fueron confirmadas para MAP por IS900-qPCR. El ADN positivo se subtipó utilizando técnicas de unidades micobacterialess repetitivas intercaladas - número variable de repeticiones en tándem (MIRU-VNTR) y técnicas de repeticiones de multilocus de secuencia corta (MLSSR) para analizar las diferencias genéticas entre los aislamientos. Resultados. El subtipado reveló dos genotipos diferentes por MIRU-VNTR (INMV 2 e INMV 36). La técnica de MLSSR se realizó para aumentar el poder discriminatorio de lo obtenido por MIRU-VNTR, pero no se observaron diferencias entre los aislamientos recuperados. Conclusiones. El presente estudio representa un enfoque importante para el conocimiento del estatus epidemiológico de MAP en la población de estudio.

ABSTRACT Objective. To determine Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in environmental samples from Colombian dairy herds. Materials and methods. Environmental samples from 25 IS900-qPCR MAP-positive dairy herds were cultured by duplicate in Herrold's egg yolk medium with mycobactin J to obtain isolates. Suspicious colonies were confirmed by MAP-IS900-qPCR. Positive DNA was sub-typed using mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) and multilocus short sequence repeats (MLSSR) techniques to analyze the genetic differences between the isolates. Results. Sub-typing revealed two different genotypes by MIRU-VNTR (INMV 2 and INMV 36). MLSSR technique was carried out to increase the discriminatory power from what was obtained by MIRU-VNTR, but no differences were observed among the recovered isolates. Conclusions. The present study represents an important approach to the knowledge on MAP epidemiological status in the study population.

Seroprevalencia a Mycobacterium avium subsp. paratuberculosis en bovinos criollos colombianos Romosinuano y costeño con cuernos / Seroprevalence to Mycobacterium aviumsubsp. paratuberculosis in the colombian creole cattle Romosinuano and costeño con cuernos

RESUMEN Objetivo. Determinar la seroprevalencia y los factores epidemiológicos asociados a Mycobacterium avium subsp Paratuberculosis (MAP) en dos razas de bovinos criollos del centro de investigación AGROSAVIA-Turipaná. Materiales y métodos. Se realizó un estudio epidemiológico de corte transversal. Fueron muestreados 848 bovinos criollos, 403 Romosinuanos y 445 costeño con cuernos (CCC); para el diagnóstico serológico de anticuerpos se utilizó la prueba Elisa Indirecta mediante el kit comercial Parachek® de Prionics. Las variables sexo, edad, raza y tipo de hato fueron los factores epidemiológicos evaluados y correlacionados con la presencia de anticuerpos contra MAP; la asociación estadística fue determinada mediante Odds Ratio y con un modelo multivariado de regresión logística, utilizando un nivel de significancia con p<0.05. Resultados. La seroprevalencia general de los bovinos criollos a MAP fue de 2.35% (IC 95%, 1.34-3.38); sin embargo, en los Romosinuano fue de 0.74% y en los CCC fue de 3.82%, siendo las diferencias estadísticamente significativas (valor p=0.003). También, empleando un análisis univariado, fueron más afectados las hembras y los animales mayores a un año. El análisis multivariado identificó como factores epidemiológicos las variables raza y sexo. Conclusiones. En las razas criollas del centro de investigación AGROSAVIA-Turipaná, la seroprevalencia a MAP fue baja; sin embargo, en la raza CCC el riesgo de contraer la enfermedad es seis veces mayor con relación a la raza Romosinuano. Más aún, se pudo evidenciar que las hembras tienen mayor riesgo de adquirir la enfermedad.

ABSTRACT Objective. Determine the seroprevalence and epidemiological factors associated with Mycobacterium avium subsp. paratuberculosis (MAP) in two Creole cattle breeds of the Turipaná research center -AGROSAVIA. Materials and methods. A cross-sectional epidemiological study was conducted,a total of 848 Creole bovine animals were sampled, 403 Romosinuano and 445 costeño con cuernos (CCC); for the serological diagnosis of antibodies, the Elisa Indirect test was used with the commercial kit Parachek®2 by Prionics. The variables sex, age, breed and herd type were the epidemiological factors evaluated and correlated with the presence of antibodies against MAP; the statistical association was established using the Odds Ratio and a multivariate logistic regression model, employing a significance level with p<0.05. Results. The general seroprevalence of the Creole cattle to MAP was 2.35% (95% CI, 1.34-3.38); however, in the Romosinuano it was 0.74% and in the CCC it was 3.82%, being this difference statistically significant (p=0.003). Furthermore, employing a univariate way analysis, females and animals older than one year of age were more affected. The multivariate analysis identified the breed and sex variables as epidemiological factors. Conclusions. In the Creole breeds of the AGROSAVIA-Turipaná research center, MAP seroprevalence was low; however, in the Costeño Con Cuernos breed, the risk of contracting the disease is six times higher than in the Romosinuano breed. Moreover, it was shown that females have a higher risk of acquiring the disease.

Detección de anticuerpos anti-Mycobacterium avium subsp. paratuberculosis (MAP) en venados silvestres en Colombia / Detection of anti-Mycobacterium aviumsubsp. paratuberculosis antibodies in wild deer in Colombia

RESUMEN Objetivo. Determinar la presencia de anticuerpos anti-Mycobacterium avium subsp. paratuberculosis (MAP) en venados de cola blanca (Odocoileus virginianus) y venados de páramo (Mazama rufína), capturados en las regiones de la Orinoquía y el Caribe en Colombia. Materiales y métodos. Un total de 44 muestras de suero sanguíneo de venados de las especies M. rufína y O. virginianus fueron colectadas en condiciones de campo entre 2014 y 2016. Se utilizó un kit comercial de ELISA para la detección de anticuerpos anti-MAP. Un animal se consideró positivo a ELISA cuando la relación muestra-positivo (S/P%) fue > 0.4, según lo recomendado por el fabricante. Resultados. El 50% (22/44) de los animales muestreados resultaron positivos, lo que corresponde a 10 hembras y 12 machos. Un 81.8% (18/22) y un 77.3% (17/22) de estos animales seropositivos fueron capturados en la región de la Orinoquía y fueron reportados como adultos, respectivamente. Conclusiones. No se sabe cómo o cuándo se introdujo MAP en la población de ciervos colombianos en las regiones de estudio. La hipótesis más plausible para explicar la presencia de anticuerpos anti-MAP en estas poblaciones silvestres es la transmisión por contacto con el ganado bovino infectado, ya que en ambas regiones estas especies comparten pasturas. Este es el primer estudio en explorar la infección por MAP en animales silvestres en Colombia. Estos hallazgos respaldan la necesidad de realizar más estudios utilizando técnicas de diagnóstico directo, y aproximaciones investigativas que permitan la definición de vínculos en la dinámica de la infección entre mamíferos silvestres y domésticos en Colombia.

ABSTRACT Objective. To determine the presence of anti-Mycobacterium avium subsp. paratuberculosis (MAP) antibodies in white-tailed deer (Odocoileus virginianus) and red brocket deer (Mazama rufína), captured in the Orinoquía and Caribbean regions in Colombia. Materials and methods. A total of 44 blood serum samples from deer of species M. rufina and O. virginianus were collected under field conditions between 2014 and 2016. An ELISA commercial kit was used to detect anti-MAP antibodies. An animal was considered ELISA-positive at a sample-to-positive ratio (S/P%) of > 0.4, as recommended by the manufacturer. Results. The 50% (22/44) of the animals were positive, corresponding to 10 females and 12 males. An 81.8% (18/22) and 77.3% (17/22) of these seropositive animals were captured in the Orinoquía region and were reported as adults, respectively. Conclusions. It is not known how or when MAP was introduced in the Colombian deer population in the study regions. The most plausible hypothesis to explain the presence of antibodies against MAP in these wild populations is transmission by contact with infected bovine cattle since, in both regions, these species share pastures. This is the first study to explore MAP infection in wild animals in Colombia. These findings support the need for further studies using different direct diagnostic techniques and research approaches that allow the definition of links in the infection dynamics between wild and domestic mammals in Colombia.

Prevalence of Mycobacterium avium subsp. paratuberculosis infection in dairy herds in Northern Antioquia (Colombia) and associated risk factors using environmental sampling.

This cross-sectional study aimed to determine Mycobacterium avium subsp. paratuberculosis (MAP) herd-level prevalence using a quantitative real-time PCR method (qPCR), performed on environmental samples. Secondly, the study aimed to explore herd-level risk factors associated with the presence of MAP in dairy herds with in-paddock milking facilities of the Northern region of the Province of Antioquia (Colombia). Study herds (n = 292) located in 61 different districts from six municipalities were randomly selected amongst 7794 dairies registered in the foot-and-mouth disease vaccination records from 2015. The sampling strategy considered a proportional allocation, both at municipality and district level. Participant herds were visited once between June and October 2016 to collect one composite environmental sample and to complete a risk assessment questionnaire. Each composite environmental sample contained material from six different sites of concentration of adult cattle and/or high traffic areas (e.g. areas surrounding waterers and feeders, areas surrounding the current mobile milking-unit places). Identification of MAP was achieved using a duplex qPCR (Bactotype MAP PCR Kit®, Qiagen). A herd was considered as MAP infected if the environmental sample was positive in the qPCR. Information about the general characteristics of the herd, management practices, and knowledge about the disease was collected using the risk-assessment questionnaire. The information on risk factors was analyzed using a multivariable logistic regression model. The apparent herd-level prevalence was 4.1% (12/292; 95% CI: 1.8-6.4). Herds with a history of mixed farming of cattle with other ruminants had higher odds of being MAP infected than herds without (OR = 3.9; 95% CI: 1.2-13.2). Our study demonstrates the MAP prevalence in dairy herds from Antioquia, Colombia and the possible relationship between MAP environmental positivity with the history of mixed farming of cattle with other susceptible ruminants.

Mycobacterium avium subsp. paratuberculosis in Colombia (1924-2016): A review / Mycobacterium avium subsp. paratuberculosis en Colombia (1924-2016): Revisión de literatura / Mycobacterium avium subsp. paratuberculosis na Colômbia (1924-2016): Revisão de literatura

Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is an acid-fast, gram-positive bacillus. MAP is the causal agent of paratuberculosis (PTB) or Johne's disease, an infectious disease affecting domestic ruminants and some wild species. Its importance as a potentially zoonotic agent due to its relation to Crohn's disease (CD) in humans is still under debate and investigation. The aim of the present systematic review is to summarize original studies on MAP carried out in Colombia since 1924, as well as to establish strengths, weaknesses, and future research opportunities in the country with emphasis on diagnosis and epidemiology. The initial search for existing publications reporting original studies on MAP, PTB, and the relationship between MAP and CD was carried out in the available databases and national libraries. After compilation of the available studies (n = 20), the relevant data was extracted (year, province of report, species studied, diagnostic tests used, study design, summary of results, and authors). Recommendations for future research opportunities on MAP in Colombia are made.

Resumen Mycobacterium avium subsp. paratuberculosis (MAP) es un bacilo ácido resistente, gram-positivo. MAP es el agente causal de la paratuberculosis (PTB) o enfermedad de Johne, una enfermedad infecciosa que afecta rumiantes domésticos y algunas especies salvajes. Su importancia como agente zoonótico, debido a su relación con la enfermedad de Crohn (CD) en humanos, está aún en debate y bajo investigación. El objetivo de la presente revisión es exponer los estudios originales sobre MAP llevados a cabo en Colombia desde 1924, así como establecer sus fortalezas, debilidades y oportunidades de investigación futura, con énfasis en los puntos de vista diagnóstico y epidemiológico. La búsqueda inicial de las publicaciones existentes sobre estudios originales realizados acerca de MAP, PTB y la relación MAP y CD fue realizada en las bases de datos disponibles y en bibliotecas nacionales. Luego de la compilación de los estudios disponibles (n = 20), los datos relevantes fueron extraídos (año, provincia de reporte, especie estudiada, prueba diagnóstica usada, diseño del estudio, resumen de resultados y autores). Se hacen recomendaciones para futuras investigaciónes de MAP en Colombia.

Resumo Mycobacterium avium subsp. paratuberculosis (MAP) é um ácido forte, bacilo gram-positivo. O MAP é o agente causador da paratuberculosis (PTB) ou doença de Johne, uma doença infecciosa que afecta os ruminantes domésticos e algumas espécies selvagens. Sua importância como um agente zoonótico por causa de sua relação com a doença de Crohn (CD) em humanos ainda está em discussão e sob investigação. O objetivo desta revisão é apresentar a estudos mapa original realizado na Colômbia desde 1924, e estabelecer pontos fortes, pontos fracos e oportunidades para futuras pesquisas no país com ênfase no diagnóstico e epidemiológico pontos de vista. A busca inicial da literatura sobre estudos originais sobre MAP, PTB e a relação MAP e CD foi feita nas bases de dados disponíveis e bibliotecas nacionais. Após a compilação dos estudos disponíveis (n = 20), os dados relevantes foram extraídos (ano, relatório província, espécies estudadas, teste de diagnóstico utilizado, desenho do estudo, os resultados resumo e autores). Foram feitas recomendações sobre futuras oportunidades de pesquisa em MAP em Colômbia.

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd / Cultivo fecal y dos métodos de PCR en materia fecal para el diagnóstico de Mycobacterium avium subsp. paratuberculosis en un hato seropositivo / Cultivo fecal e dois métodos de PCR em matéria fecal para o diagnóstico de Mycobacterium avium subsp. paratuberculosis em um rebanho soropositivo

Abstract Background: paratuberculosis is a slow-developing infectious disease, characterized by chronic granulomatous enterocolitis. This disease has a variable incubation period from 6 months to over 15 years, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Its detection by direct and indirect diagnostic techniques has been of special interest. Objective: to report the diagnosis and detection of MAP using several diagnostic tests in a herd of the Northern region of Antioquia, Colombia. Methods: serum samples from the study herd were analyzed, using a commercial ELISA (enzyme-linked immunosorbent assay) kit. Fecal samples were cultured by duplicate using Herrold´s egg yolk medium (HEYM), and analyzed by an end- point IS900-specific nested PCR protocol, and a commercial F57-real-time PCR kit. Results: eight out of 27 serum samples in the study herd resulted ELISA-positive. None of fecal samples resulted positive to HEYM culture by duplicate and none were found to be positive by F57-real-time PCR. Seven of the 27 fecal samples were found to be positive by end-point IS900-specific nested PCR. Agreement was found between ELISA and end-point IS900-specific nested PCR in one of the animals. Conclusion: the present study gives information about the agreement between direct and indirect MAP-detection techniques, using different matrixes from animals under the same husbandry conditions.

Resumen Antecedentes: la paratuberculosis es una enfermedad infecciosa de desarrollo lento, caracterizada por una enterocolitis granulomatosa crónica. Esta enfermedad tiene un periodo de incubación que varía entre los 6 meses hasta por más de 15 años, y es causada por Mycobacterium avium subsp. paratuberculosis (MAP). Su detección por técnicas diagnósticas directas e indirectas ha sido de interés especial. Objetivo: reportar el diagnóstico y detección de MAP utilizando varias técnicas diagnósticas en un hato de la región norte de Antioquia, Colombia. Métodos: se analizaron las muestras de suero del hato de estudio utilizando un kit comercial de ELISA (enzyme-linked immunosorbent assay). Las muestras de materia fecal fueron cultivadas por duplicado en Herrold´s egg yolk medium (HEYM), y analizadas mediante un protocolo de PCR anidado específico de IS900 y un kit comercial de PCR en tiempo real para F57. Resultados: ocho de las 27 muestras de suero resultaron positivas por ELISA. Ninguna de las muestras de materia fecal resultó positiva al cultivo en HEYM por duplicado ni por PCR en tiempo real para F57. Siete de las 27 muestras de materia fecal resultaron positivas a PCR anidado específico de IS900. Se encontró concordancia entre el resultado de ELISA y de PCR anidado específico de IS900 en uno de los animales. Conclusión: el presente estudio brinda información acerca de la concordancia entre técnicas directas e indirectas de detección de MAP, utilizando diferentes matrices a partir de animales bajo las mismas condiciones de manejo.

Resumo Antecedentes: a paratuberculosis é uma doença infecciosa de evolução lenta, caracterizada por uma enterocolite granulomatosa crônica. Esta doença tem um período de incubação que varia de 6 meses a 15 anos e é causada pelo Mycobacterium avium subsp. paratuberculosis (MAP). Sua detecção por técnicas de diagnóstico diretos e indiretos tem sido de especial interesse. Objetivo: reportar o diagnóstico e a detecção de MAP utilizando várias técnicas de diagnóstico em um rebanho na região norte de Antióquia, Colômbia. Métodos: foram analisadas amostras de soro do rebanho utilizando um kit comercial de ELISA (enzyme- linked immunosorbent assay). As amostras de fezes foram cultivadas em duplicado em Herrold´s egg yolk medium (HEYM) e analisadas utilizando um protocolo de PCR aninhada específico de IS900 e um kit de PCR em tempo real comercial para F57. Resultados: oito das 27 amostras de soro foram positivas para ELISA. Nenhuma das amostras testadas na cultura de fezes HEYM duplicado foram positivas ou na PCR em tempo real para F57. Sete das 27 amostras de fezes foram positivas na PCR aninhada específica para IS900. Foi encontrada concordância entre o resultado de ELISA e PCR aninhada específica para IS900 em um animal. Conclusão: este estudo fornece informações sobre a correlação entre técnicas de detecção direta e indireta do MAP, utilizando diferentes matrizes de animais sob as mesmas condições de condução.

Evaluation of two in-house immunoenzymatic tests to serodiagnose subclinical paratuberculisis in a sheep flock in Mexicali valley, Mexico.

Paratuberculosis (PTB) or Johne's disease is a common ruminant infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, two MAP antigens were compared for their diagnostic utility to detect subclinical PTB in a sheep flock in Mexicali, Mexico. Sheep (n = 31) without clinical signs but positive on a direct fecal-polymerase chain reaction were tested with two preabsorbed in-house enzyme linked immunosorbent assays (ELISAs) using: (1) an ethanol-extracted surface lipid antigen (EVELISA) and (2) a protoplasmic antigen (ELISA-PPA). Sensitivities of the EVELISA and ELISA-PPA were 84% (95% CI; 66-95%) and 29% (95% CI; 14-48%), respectively. The EVELISA test could be a fast and effective way to identify subclinical ovine PTB for severely affected flocks.

Paratuberculosis in buffaloes in Northeast Brazil.

Several farms in the Northeast of Brazil were investigated for Mycobacterium avium subsp. paratuberculosis infection in order to identify the occurrence of paratuberculosis in buffaloes. Samples were obtained from 17 farms, two slaughter houses, and a quarantine area in the Northeast. About 15,000 buffaloes of the Murrah, Mediterranean, and Jafarabadi breed as well as their crossbreeds were evaluated for meat, dairy, and mixed farms with semi-intensive or extensive breeding practices. For diagnostic purposes, postmortem and histopathological examination, including Ziehl-Neelsen test of fecal smears and scraped intestinal mucosa were performed. PCR was applied for fecal samples, mesenteric lymph nodes, and intestines. Six Johne's disease-positive farms, which together with those previously identified, indicate that the disease is spread through the Brazilian Northeast, similar to what occurs in cattle herds in other regions of the country. The increase in prevalence of paratuberculosis is a consequence of introduction of animals from other regions without adequate veterinary assistance and due to the little official attention paid to this initially silent and chronic disease.

Milk yield and lactation stage are associated with positive results to ELISA for Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia: a preliminary study.

Paratuberculosis is a slow-developing infectious disease characterized by chronic granulomatous enterocolitis. This disease has a variable incubation period from 6 months to over 15 years and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Some studies have been conducted in cattle during the last decades in Colombia. However, those studies were designed using relatively small populations and were not aimed to establish prevalence. This study aimed to determine the MAP seroprevalence in selected dairy herds and to explore risk factors associated with the serology results. Serum samples and related data were collected from 696 randomly selected bovines in 28 dairy herds located in 12 different districts in one of the main dairy municipalities in Colombia (San Pedro de los Milagros). The samples were analyzed using a commercial ELISA kit. The information on risk factors was analyzed using a logistic regression. The apparent seroprevalence was 3.6 % (1/28) at the herd level and 2 % (14/696) at the animal level. The number of days in milk production between 100 and 200 days, and over 200 days as well as the daily milk production between 20 and 40 L/cow, and over 40 L/cow were associated with MAP seropositivity with odds ratios of 4.42, 3.45, 2.53, and 20.38, respectively. This study demonstrates the MAP seroprevalence in dairy herds from Antioquia, Colombia and the possible relationship between MAP seropositivity, milk yield, and lactation stage.

Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention.

Diagnóstico de paratuberculose por biópsia retal em búfalos / Rectal biopsy for the diagnosis of paratuberculosis in buffaloes

Foram realizadas biópsias retais de 140 búfalos, machos e fêmeas, das raças Murrah e mestiços de Murrah com Mediterrâneo, com idade acima de três anos, em uma propriedade no município de São Mateus, Maranhão, Brasil. Adicionalmente foram realizadas necropsias de 11 búfalos, para realizar um estudo comparativo entre os achados das biópsias retais e de tecidos de íleo e linfonodo mesentérico. A propriedade apresentava histórico de animais com emagrecimento progressivo e diarreia não responsiva a antimicrobianos. Os búfalos apresentavam sinais clínicos caracterizados por diarreia, estado nutricional regular a ruim, desidratação e edema submandibular. Nas biópsias retais seis búfalos apresentaram lesões sugestivas da paratuberculose na Hematoxilina-Eosina (HE), sendo estas caracterizadas por inflamação granulomatosa multifocal moderada na lâmina própria com macrófagos epitelioides. Em quatro animais foram observadas adicionalmente células gigantes do tipo Langhans. Em 15 búfalos foi observado infiltrado linfocitário multifocal leve na lâmina própria. Pela coloração de Ziehl-Neelsen (ZN), 4,3% (6/140) apresentaram bacilos álcool-ácido resistentes (BAAR) e na PCR em tempo real (qPCR), 5,71% (7/140) tiveram amplificação do material genético. Foram necropsiados 11 búfalos, à necropsia foram observados aumento de linfonodos mesentéricos com áreas esbranquiçadas na superfície de corte; intestino delgado e grosso com dobras transversais evidentes, mucosa espessada e irregular, de aspecto reticulado, placas de Peyer evidentes e conteúdo líquido e marrom. Ainda se viam áreas espessadas em torno da válvula ileocecal e vasos linfáticos evidentes. As lesões histológicas localizadas no intestino delgado e linfonodos mesentéricos de quatro búfalos foram compatíveis com lesões já descritas na literatura, e apresentaram BAAR e amplificação de material genético na qPCR. A concordância entre a biópsia retal e a análise dos tecidos de íleo e linfonodo mesentérico, segundo...

Paratuberculosis in a herd of buffaloes was studied in the municipality of São Mateus, Maranhão, Brazil. Rectal biopsies were performed in 140 male and female Murrah, Mediterranean and crossbreed buffaloes older than 3 years. Postmortem examination of 11 buffaloes was performed to compare the rectal biopsies with possible lesions in mesenteric nodes and the intestine. The history of the herd and clinical examination revealed progressive weight loss and non-responsive antimicrobial diarrhea, dehydration and submandibular edema. Rectal biopsies showed in six buffaloes microscopically suggestive lesions for paratuberculosis through hematoxilin-eosin staining (HE), characterized by moderate multifocal granulomatous enteritis with epithelioid cell infiltration. In four buffaloes Langhans giant cells were found. In 15 buffaloes lymphocytic infiltrate was observed in the lamina propria of the large intestine. Ziehl-Neelsen staining (ZN) revealed in 4.3% (6/140) acid-fast bacilli in the rectal mucosa. Real time PCR amplified to 5.71% (7/140) Mycobacterium avium subsp. paratuberculosis (Map) DNA. 11 buffalos were submitted to postmortem examination, gross examination revealed augmented mesenteric nodes with whitish areas in the cut surface. The mucosa of the small intestine was irregular and thickened, with evident traverse folds and Peyer plates. The brownish intestinal content was fluid, the ileocecal valve area thickened and edematous with evident lymphatic vessels. Histological lesions in the mesenteric lymph node and small intestine four buffalo were compatible with those already described in the literature, and presented acid-fast bacilli by ZN staining and amplification of Map genetic material in qPCR. The concordance between the rectal biopsy and the postmortem samples was in agreement with the Kappa test (K=0.792) and was considered substantial or high. The rectal biopsy showed to be promising and can be used by practitioners, together with other...

Diagnóstico de paratuberculose por biópsia retal em búfalos / Rectal biopsy for the diagnosis of paratuberculosis in buffaloes

Foram realizadas biópsias retais de 140 búfalos, machos e fêmeas, das raças Murrah e mestiços de Murrah com Mediterrâneo, com idade acima de três anos, em uma propriedade no município de São Mateus, Maranhão, Brasil. Adicionalmente foram realizadas necropsias de 11 búfalos, para realizar um estudo comparativo entre os achados das biópsias retais e de tecidos de íleo e linfonodo mesentérico. A propriedade apresentava histórico de animais com emagrecimento progressivo e diarreia não responsiva a antimicrobianos. Os búfalos apresentavam sinais clínicos caracterizados por diarreia, estado nutricional regular a ruim, desidratação e edema submandibular. Nas biópsias retais seis búfalos apresentaram lesões sugestivas da paratuberculose na Hematoxilina-Eosina (HE), sendo estas caracterizadas por inflamação granulomatosa multifocal moderada na lâmina própria com macrófagos epitelioides. Em quatro animais foram observadas adicionalmente células gigantes do tipo Langhans. Em 15 búfalos foi observado infiltrado linfocitário multifocal leve na lâmina própria. Pela coloração de Ziehl-Neelsen (ZN), 4,3% (6/140) apresentaram bacilos álcool-ácido resistentes (BAAR) e na PCR em tempo real (qPCR), 5,71% (7/140) tiveram amplificação do material genético. Foram necropsiados 11 búfalos, à necropsia foram observados aumento de linfonodos mesentéricos com áreas esbranquiçadas na superfície de corte; intestino delgado e grosso com dobras transversais evidentes, mucosa espessada e irregular, de aspecto reticulado, placas de Peyer evidentes e conteúdo líquido e marrom. Ainda se viam áreas espessadas em torno da válvula ileocecal e vasos linfáticos evidentes. As lesões histológicas localizadas no intestino delgado e linfonodos mesentéricos de quatro búfalos foram compatíveis com lesões já descritas na literatura, e apresentaram BAAR e amplificação de material genético na qPCR. A concordância entre a biópsia retal e a análise dos tecidos de íleo e linfonodo mesentérico, segundo...(AU)

Paratuberculosis in a herd of buffaloes was studied in the municipality of São Mateus, Maranhão, Brazil. Rectal biopsies were performed in 140 male and female Murrah, Mediterranean and crossbreed buffaloes older than 3 years. Postmortem examination of 11 buffaloes was performed to compare the rectal biopsies with possible lesions in mesenteric nodes and the intestine. The history of the herd and clinical examination revealed progressive weight loss and non-responsive antimicrobial diarrhea, dehydration and submandibular edema. Rectal biopsies showed in six buffaloes microscopically suggestive lesions for paratuberculosis through hematoxilin-eosin staining (HE), characterized by moderate multifocal granulomatous enteritis with epithelioid cell infiltration. In four buffaloes Langhans giant cells were found. In 15 buffaloes lymphocytic infiltrate was observed in the lamina propria of the large intestine. Ziehl-Neelsen staining (ZN) revealed in 4.3% (6/140) acid-fast bacilli in the rectal mucosa. Real time PCR amplified to 5.71% (7/140) Mycobacterium avium subsp. paratuberculosis (Map) DNA. 11 buffalos were submitted to postmortem examination, gross examination revealed augmented mesenteric nodes with whitish areas in the cut surface. The mucosa of the small intestine was irregular and thickened, with evident traverse folds and Peyer plates. The brownish intestinal content was fluid, the ileocecal valve area thickened and edematous with evident lymphatic vessels. Histological lesions in the mesenteric lymph node and small intestine four buffalo were compatible with those already described in the literature, and presented acid-fast bacilli by ZN staining and amplification of Map genetic material in qPCR. The concordance between the rectal biopsy and the postmortem samples was in agreement with the Kappa test (K=0.792) and was considered substantial or high. The rectal biopsy showed to be promising and can be used by practitioners, together with other...(AU)

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice / Expressão de citocinas e detecção molecular de Mycobacterium avium subspecies paratuberculosis em órgãos de camundongos infectados experimentalmente

Mycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Mycobacterium avium subespécie paratuberculosis (MAP) pode infectar ruminantes e permanecer subclínica por longos períodos nos rebanhos. A identificação de órgãos mais susceptíveis à infecção e a avaliação da expressão das citocinas no local da infecção são importantes para compreender a patogênese de MAP. Neste estudo foi avaliada a probabilidade de detecção de DNA de MAP e a expressão de citocinas em órgãos de camundongos C57BL/6 infectados por via intraperitoneal durante 120 dias. Dentre os órgãos avaliados, o baço (85%), cólon (75%) e fígado (60%) tiveram as maiores frequências de positividade. Quando comparadas essas frequências entre os órgãos, verificou-se que o baço teve 1,54 vezes mais probabilidade de ser positivo em relação ao íleo, e 2,0 vezes mais probabilidade em relação às placas de Peyer. Além disso, aos 60 dias pós infecção, o baço e o fígado foram responsáveis pela maior expressão de IFN-γ e o íleo pela TNF-α e IL-4. Os resultados indicam que o baço é o melhor órgão para avaliar uma infecção experimental por MAP, principalmente nos períodos iniciais da infecção. Além disso, demonstrou que o baço, fígado e íleo têm importância direta na resposta inflamatória de modelos experimentais.