RESUMO
Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund's adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos , Citocinas , Modelos Animais de Doenças , Imunização Secundária , Imunoglobulina G , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Mycobacterium avium subsp. paratuberculosis/imunologia , Imunização Secundária/métodos , Camundongos , Paratuberculose/prevenção & controle , Paratuberculose/imunologia , Imunoglobulina G/sangue , Citocinas/metabolismo , Feminino , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Camundongos Endogâmicos BALB C , Vaccinia virus/imunologia , Vaccinia virus/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Imunidade Celular/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologiaRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.
Assuntos
Doenças dos Bovinos , Ensaio de Imunoadsorção Enzimática , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Sensibilidade e Especificidade , Bovinos , Animais , Paratuberculose/diagnóstico , Paratuberculose/sangue , Paratuberculose/microbiologia , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/sangue , Doenças dos Bovinos/microbiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Testes Sorológicos/veterinária , Testes Sorológicos/métodos , ArgentinaRESUMO
Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942 ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage.
Assuntos
Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/microbiologia , Cobre , Antioxidantes , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis. This pathogen is able to survive adverse environmental conditions, including the pasteurization process. Copper, a well-studied metal, is considered an important antibacterial tool, since it has been shown to inactivate even MAP in treated milk through unknown mechanisms. The aim of the present study is to show the effect of copper ions, and reactive oxygen species (ROS) generated in response to oxidative stress, on the damage to MAP DNA when exposed to a copper ion challenge in cow's milk. METHODOLOGY: Spiked milk with different MAP bacterial loads was supplemented with blocking agents. These were either the copper chelators ethylenediaminetetraacetic acid (EDTA) and batocuproin (BCS) or the ROS quenchers D-mannitol, gallic acid and quercetin. The DNA protection, MAP viability and ROS production generated after exposure to a copper challenge were then measured. RESULTS: In a bacterial load of 104 cells mL-1, blocking effects by both the copper chelators and all the ROS quenchers offered significant protection to MAP DNA. In a concentration of 102 cells mL-1, only D-mannitol and a mix of quenchers significantly protected the viability of the bacteria, and only at a concentration of 106 cells mL-1 was there a lower production of ROS when supplementing milk with gallic acid, quercetin and the mix of quenchers. CONCLUSION: Based on these findings, it may be concluded that MAP DNA damage can be attributed to the combined effect of the direct copper ions and ROS generated. Nevertheless, taking into account the antioxidant environment that milk provides, the direct effect of copper could play a prominent role.
RESUMO
BACKGROUND: Scientific evidence is scarce for the antimicrobial effect of copper on bacteria characterized as more resistant. Using Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant microorganism, as a pathogen model, copper ion treatment has shown a significant bactericidal effect; however, the sustainability of MAP against copper toxicity was also reported in several studies. Accordingly, the present study aimed to evaluate the impacts of copper on MAP. METHODOLOGY: This study considered physicochemical properties and copper concentration in a buffer since it could modulate MAP response during the application of copper treatment. RESULTS: Despite the efficacy of copper ions in significantly reducing the MAP load in Phosphate Buffered Saline, some MAP cells were able to survive. The copper concentration generated by the copper ion treatment device increased significantly with increasing exposure times. MAP bacterial load decreased significantly when treated with copper ions as the exposure times increased. An increase in pH decreased oxygen consumption, and an increase in conductivity was reported after treatment application. CONCLUSIONS: Even with higher concentrations of copper, the efficacy of MAP control was not complete. The concentration of copper must be a key element in achieving control of highly resistant microorganisms.
RESUMO
The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.
Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Virulência/genética , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Doenças dos Bovinos/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Animais , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/veterinária , Bovinos , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
RESUMEN La Paratuberculosis Bovina (PTB) o Enfermedad de Johne, es una infección del tracto gastrointestinal causada por Mycobacterium avium, subespecie paratuberculosis (Map), que se caracteriza por generar enteritis granulomatosa crónica y linfadenitis en rumiantes. La preocupación más relevante en relación con la importancia de la PTB es su posible vínculo con la Enfermedad de Crohn (EC) en humanos, sin embargo, esta asociación aún está bajo investigación. Se determinó la seroprevalencia de PTB en el municipio de Sogamoso (Boyacá), donde se recolectaron 604 muestras de sangre, cuyo suero fue procesado mediante la técnica de ELISA indirecta con el kit comercial PARACHEK® 2 KIT (Prionics, Suiza). La seroprevalencia fue de 10,9% (66/604), donde el grupo etario de 2 a 3 años y la raza Jersey fueron los de mayor seroprevalencia. Se encontró asociación estadística significativa (p≤0,05) entre la edad de los individuos evaluados y el suministro de concentrado. La seroprevalencia encontrada indica que se está produciendo una transmisión activa de la enfermedad y que las medidas de control disponibles no están siendo llevadas a cabo o no son lo suficientemente efectivas.
ABSTRACT Bovine Paratuberculosis (BPT), or Johne's Disease, is an infection of the gastrointestinal tract caused by Mycobacterium avium, subspecies paratuberculosis (Map), which is characterized by chronic granulomatous enteritis and lymphadenitis in ruminants. The most relevant concern regarding the importance of BPT is its possible link to Crohn's disease (CD) in humans, however this association is still under investigation. The seroprevalence of BPT was determined in the municipality of Sogamoso (Boyacá), where 604 blood samples were collected, their serum was processed by the indirect ELISA technique with the commercial PARACHEK® 2 KIT (Prionics, Switzerland), following the manufacturer's instructions. The seroprevalence was 10,9% (66/604), with the 2 to 3 years age group and the Jersey breed having the highest seroprevalence. A significant statistical association was found (p≤0,05) with the age of the individuals tested and the supply of concentrate. The seroprevalence found indicates that active transmission of the disease is taking place, and that the available control measures are not being carried out or are not effective enough.
RESUMO A Paratuberculose Bovina (PTB) ou Doença de Johne é uma infecção do trato gastrointestinal causada pela Mycobacterium avium subespécie paratuberculosis (Map), caracterizada por gerar enterite granulomatosa crónica e linfadenite em ruminantes. A preocupação mais relevante em relação à importância do PTB é seu possível vínculo com a Doença de Crohn (DC) em seres humanos, no entanto, essa associação ainda está sob investigação. A soroprevalência do PTB foi determinada no município de Sogamoso (Boyacá), onde foram coletadas 604 amostras de sangue, cujo soro foi processado pela técnica ELISA indireta com o kit comercial PARACHEK® 2 KIT (Prionics, Suíça). A soroprevalência foi de 10,9% (66/604), onde a faixa etária de 2 a 3 anos e a raça Jersey foram as que apresentaram maior soroprevalência. Foi encontrada associação estatisticamente significante (p≤0,05) com a idade dos indivíduos avaliados e o suprimento de concentrado. A soroprevalência encontrada indica que a transmissão ativa da doença está ocorrendo e que as medidas de controle disponíveis não estão sendo realizadas ou não são eficazes o suficiente.
RESUMO
The objective of this study was to estimate the performance of the peptide magnetic separation PCR test (PMS-PCR) for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) in sub-clinically infected dairy cattle. Twenty-one herds were randomly selected from a source population of 131 commercial dairy herds with a known history of MAP infection, located in the De Los Rios and De Los Lagos regions, in southern Chile. In the selected herds, all milking cows with ≥2 parities and without any clinical signs were sampled, collecting feces and blood-serum samples. The PMS-PCR test was used to analyze the fecal samples, while serum samples were analyzed using a commercial ELISA kit. A Bayesian latent class model was used to estimate the sensitivity (Se) and specificity (Sp) of the diagnostic tests. A total of 1381 animals were sampled in the 21 selected dairy herds, with an average sample size of 65 animals per herd (range 10-721). The PMS-PCR test had a greater Se than the ELISA test, with a median of 85.5 % (posterior probability interval (PPI) 95 %: 79.3-91.0%), while the ELISA test presented a median of 21.7 % (95 % PPI: 18.3-25.4%). On the other hand, the ELISA test had a better Sp than the PMS-PCR test, with a median of 97.7 % (95 % PPI: 96.6-98.5%), whereas PMS-PCR presented a median of 90.8 % (95 % PPI: 88.3-93.9%). Model results showed that PMS-PCR has a better Se than all available tests for MAP diagnosis in subclinical animals. However, this test should be used with care in herds with high infection rates, where a high MAP environmental load is expected, potentially increasing the frequency of false positive cases due to the pass-through phenomenon.
Assuntos
Doenças dos Bovinos/diagnóstico , Testes Diagnósticos de Rotina/veterinária , Separação Imunomagnética/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Animais , Infecções Assintomáticas/epidemiologia , Teorema de Bayes , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Chile/epidemiologia , Indústria de Laticínios , Testes Diagnósticos de Rotina/instrumentação , Análise de Classes Latentes , Paratuberculose/epidemiologia , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/métodos , Prevalência , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic infectious contagious disease of the intestinal tract of ruminants that are also associated with Crohn's disease in humans. The existence of paratuberculosis in Ecuador is virtually unknown; hence, the present study was performed to gain insight into the prevalence of this disease. Methods: Three dairy cattle farms in different geographic regions in Ecuador were investigated for the infection with MAP, and 600 blood samples, 200 of each cattle herd, were processed with an indirect enzyme-linked immunosorbent assay. Fecal samples of the seropositive cows were processed for culture on modified Löwenstein-Jensen medium. Results: One hundred and fifty bovines (25%) resulted seropositive and we confirmed with culture the presence of MAP in 4.7% (7/150) of the seropositive cows. Approximately 20% of the fecal samples of seropositive cows yielded nontuberculous mycobacteria (NTM) species including M. avium subsp. avium, a NTM species closely related to MAP. Conclusions: The seroprevalence of paratuberculosis in this first study for Ecuador is high (25%). We discuss a possible interference of NTM species, isolated from fecal samples, with the diagnosis of paratuberculosis. With this report, a baseline study, we confirm for the first time the presence of paratuberculosis in Ecuador, and we provide the necessary information for future studies and control of this disease.
Assuntos
Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/epidemiologia , Animais , Indústria de Laticínios , Equador/epidemiologia , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Feminino , Prevalência , Estudos SoroepidemiológicosRESUMO
Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8 weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.
Assuntos
Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Animais , Brasil/epidemiologia , Búfalos/microbiologia , Bovinos , Doenças dos Bovinos/epidemiologia , DNA Bacteriano/química , Fezes/microbiologia , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The study objective was to identify risk factors associated to: i) the infection by Mycobacterium avium subsp. paratuberculosis (MAP), and ii) paratuberculosis clinical incidence in Chilean dairy herds. A random sample of forty herds with previous history of MAP infection was selected. At herd level, all lactating cows were tested using a commercial ELISA kit. On the sampling date, a questionnaire gathering information on herd demographics, husbandry practices, and biosecurity measures was applied. Additionally, the farm manager/owner was surveyed regarding the number of paratuberculosis compatible clinical cases (CCC) in the last 12 months. Two Bayesian generalized linear mixed effect models were used to evaluate the association between the questionnaire data, and the proportion of truly infected animals (model 1) or the number of CCC (model 2). A total of 4963 animals were sampled with an average apparent prevalence of 6.3 % (95 % confidence interval (4.0-8.0%). All sampled herds presented seropositive animals. Forty eight percent of the herds did not observe any CCC in the last year. Although, among those herd that did report CCC, a median of two cases per year was estimated. Model outputs showed that the proportion of truly infected animals and CCC reporting rates are associated to management practices. Specifically, positive associations were observed for feeding of calves exclusively with milk replacer, and the distance between the milking parlor and the calves' barn. Additionally, CCC reporting rates were higher in farms that recently purchased animals, and where the distance between the milking parlor and the calves' barn was less than 30â¯m.
Assuntos
Doenças dos Bovinos/epidemiologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/virologia , Chile/epidemiologia , Feminino , Incidência , Paratuberculose/virologia , Prevalência , Fatores de RiscoRESUMO
Background: Bovine tuberculosis control programs are based on a standard diagnostic method, the intradermal test with purified protein derivatives, which is used to identify and eliminate diseased animals. Currently none of the tests available allow complete differentiation between infected and uninfected animals. The main limitations of the tests available are related to diagnostic sensitivity and specificity, which results in false positive reactions due to the existence of cross infections, and also false negative, inherent to the state of energy of some animals. The aim of this work was to study the intercurrence of paratuberculosis in tuberculosis reactive cattle by the comparative cervical test. Materials, Methods & Results: Three hundred and thirty four cattle were evaluated using the comparative cervical test (CCT) and serology for tuberculosis (TB) and paratuberculosis (PTB) ELISA IDEXX®. All of the animals testing positive by CCT were euthanized and necropsied. Fragments of lymph node, lung and intestine were collected and analyzed using histopathological techniques, with staining by Hematoxylin-Eosin (HE). Samples of lung and lymph nodes (retropharyngeal, submandibular, cervical and mediastinal) of the animals testing positive by CCT were evaluated using qPRC for M. bovis, and intestinal and mesenteric lymph nodes using PCR for PTB. Of the 334 cattle evaluated using the comparative cervical test, 16 were considered positive. No lesions suggestive of tuberculosis were found in the macroscopic inspection of the carcasses. The most evident anatomical and pathological finding was a thickening of intestinal mucosa, found in 12 of the 16 cattle submitted to necropsy. No microscopic lesions suggestive of TB were identified nor was the presence of M. bovis detected by qPCR. The main histopathological findings were observed in the small intestine and mesenteric lymph nodes and identified as enteritis, lymphangitis...
Assuntos
Animais , Bovinos , Paratuberculose/diagnóstico , Paratuberculose/epidemiologia , Teste Tuberculínico/métodos , Teste Tuberculínico/veterinária , Mycobacterium avium subsp. paratuberculosis , Mycobacterium bovisRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.
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Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Citocinas/metabolismo , Macrófagos/microbiologia , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Paratuberculose/microbiologia , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Bovinos , Linhagem Celular , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/tratamento farmacológico , Paratuberculose/imunologia , CatelicidinasRESUMO
The Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (Johne's disease), a systemic and chronic inflammation of intestine that affects bovine, small ruminants like goat and sheep. The disease has a greater economic importance in cattle and in small ruminants. But its effective control is impeded due to lack of rapid and accurate diagnostics. The present study is aimed at developing a LAMP-coupled lateral flow device (LFD) for rapid detection of paratuberculosis in livestock animal species such as cattle and in small ruminants at resource-limited areas. LAMP primers with biotin and FITC end tags were designed for IS900 gene specific for MAP. To determine sensitivity of LAMP assay, 10-fold serial dilutions were made from 10 ng/µl MAP stock DNA and were compared with PCR. The detection limits of LAMP-coupled LFD were defined and reactions were repeated for reproducibility. The specificity was evaluated using other infectious bacteria such as M. bovis, M. tuberculosis, Brucella abortus, Leptospira interrogan, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogens, and Staphylococcus aureus. A total of 95 samples turned positive for LAMP-coupled LFD out of 389 fecal samples. All the cultural-positive and PCR-positive samples showed positive in LAMP-coupled LFD. Nine samples with negative cultures turned positive in LAMP assay. The overall sensitivity and specificity of the LAMP-coupled LFD assays were 100% and 97.02% respectively in comparison with the culture as the gold standard method. The sensitivity detection limit of developed assay was 10 fg/µl and specificity was 100%. This assay successfully detected MAP not only by using bacterial DNA but also in clinical fecal samples. The clear band formation at control and test positions was observed on LAMP-coupled LFD. The developed assay is a simple, rapid, easy to perform, and is very useful in early diagnosis of Mycobacterium avium subsp. paratuberculosis at point of care resource-limited areas.
Assuntos
Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Paratuberculose/microbiologia , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Primers do DNA/genética , Doenças das Cabras/diagnóstico , Doenças das Cabras/microbiologia , Cabras , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Paratuberculose/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/microbiologiaRESUMO
The consolidation of the dairy industry, with increase in sizes, density and productivity of the herds, was associated to unprecedented bovine tuberculosis (bTB) prevalence levels in dairy herds in Uruguay, where Johne's disease (JD), another mycobacterial disease, is also prevalent. Here, we aimed to characterize the association between bTB- and JD-diagnostic results in two heavily bTB- and JD-coinfected dairy herds. Results from bTB-intradermal tests and JD-ELISA in 686 cows indicated a significantly (P < 0.001) higher frequency of bTB-positive animals in the JD-positive population, in which a significantly lower agreement between the caudal and cervical comparative intradermal tests was observed, compared to the JD-negative population. These findings suggest a significant association between the detection of these mycobacterial diseases, that may affect the performance of the routine bTB diagnostic tests performed in dairy herds in Uruguay.
RESUMO
BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. RESULTS: Moderate numbers (103 CFU ml-1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. CONCLUSIONS: The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.
Assuntos
Cobre/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Soluções Tampão , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mycobacterium avium subsp. paratuberculosis/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.
Assuntos
Vacinas Bacterianas/imunologia , Imunidade Inata , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Paratuberculose/prevenção & controle , Animais , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium avium subsp. paratuberculosis/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
This study aimed to estimate the distributions of the within-herd true prevalence (TP) and the annual clinical incidence proportion (CIp) of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds in Chile. Forty two commercial herds with antecedents of MAP infection were randomly selected to participate in the study. In small herds (≤30 cows), serum samples were collected from all animals present. Whereas, in larger herds, milk or serum samples were collected from all milking cows with 2 or more parities. Samples were analysed using the Pourquier® ELISA PARATUBERCULOSIS (Insitute Pourquier, France) test. Moreover, a questionnaire gathering information on management practices and the frequency of clinical cases, compatible with paratuberculosis (in the previous 12 months), was applied on the sampling date. A Bayesian latent class analysis was used to obtain TP and clinical incidence posterior distributions. The model adjusts for uncertainty in test sensitivity (serum or milk) and specificity, and prior TP & CIp estimates. A total of 4963 animals were tested, with an average contribution of 124 samples per herd. A mean apparent prevalence of 6.3% (95% confidence interval: 4.0-8.0%) was observed. Model outputs indicated an overall TP posterior distribution, across herds, with a median of 13.1% (95% posterior probability interval (PPI); 3.2-38.1%). A high TP variability was observed between herds. CIp presented a posterior median of 1.1% (95% PPI; 0.2-4.6%). Model results complement information missing from previously conducted epidemiological studies in the sector, and they could be used for further assessment of the disease impact and planning of control programs.
Assuntos
Doenças dos Bovinos/epidemiologia , Paratuberculose/epidemiologia , Animais , Teorema de Bayes , Bovinos , Chile/epidemiologia , Indústria de Laticínios , Ensaio de Imunoadsorção Enzimática , Feminino , Incidência , Mycobacterium avium subsp. paratuberculosis , PrevalênciaRESUMO
LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.