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1.
Sci Total Environ ; 587-588: 466-472, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28258755

RESUMEN

The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans, food-producing animals and food world-wide. However, the occurrence and impact of these so-called extended spectrum cephalosporinase (ESC)-producing Enterobacteriaceae in aquatic environments are poorly documented. This study investigated the occurrence, concentrations and characteristics of ESC-producing E. coli (ESC-Ec) in samples of surface water collected at five Swedish water treatment plants that normally have relatively high prevalence and concentration of E. coli in surface water. ESC-Ec was found in 27 of 98 surface water samples analysed. All but two positive samples were collected at two of the water treatment plants studied. The ESC-Ec concentration, 1-3cfu/100mL, represented approximately 4% of the total amount of E. coli in the respective surface water sample. In total, 74% of the isolates were multi-resistant, but no isolate was resistant to carbapenems. Six types of ESBL/pAmpC genes were found in the 27 E. coli isolates obtained from the positive samples, of which four (blaCTX-M-15, blaCMY-2, blaCTX-M-1 and blaCTX-M-14) were found during the whole sampling period, in samples taken at more than one water treatment plant. In addition, the genes were situated on various types of plasmids and most E. coli isolates were not closely related with regard to MLST types. The combinations of ESBL/pAmpC genes, plasmids and E. coli isolates were generally similar to those found previously in healthy and sick individuals in Sweden. In conclusion, the occurrence of ESC-Ec in Swedish surface water shows that resistant bacteria of clinical concern are present in aquatic environments even in a low-prevalence country such as Sweden.


Asunto(s)
Cefalosporinasa/metabolismo , Agua Potable/microbiología , Monitoreo del Ambiente , Microbiología del Agua , Proteínas Bacterianas/metabolismo , Escherichia coli , Tipificación de Secuencias Multilocus , Suecia , beta-Lactamasas/metabolismo
2.
J Antimicrob Chemother ; 72(2): 582-588, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27798205

RESUMEN

OBJECTIVES: Community carriage of ESBL-producing Escherichia coli (EPE) is common worldwide and there is a need to understand the connection between carriage and infection. We compared the molecular characteristics of EPE among Swedish community carriers with those of EPE causing invasive infections. METHODS: We collected 2134 faecal samples from randomly selected Swedish inhabitants and examined them for the presence of EPE. All participating volunteers answered a questionnaire about putative risk factors for EPE carriage. Suspected EPE isolates (n = 418) from patients with bloodstream infection (BSI) were collected from Swedish laboratories. Isolates were genotypically and phenotypically characterized. RESULTS: Our results show that the EPE population found in carriers generally had lower pathogenicity compared with the isolates from BSIs, since carriers had a lower proportion of E. coli belonging to phylogroup B2, ST131 and ST131 subclone H30-Rx. Isolates from carriers also had lower levels of multiresistance. The Swedish carriage rate of EPE was 4.7% (101/2134) among healthy volunteers. Risk factors associated with carriage were travel to countries in Asia (OR = 3.6, 95% CI = 1.4-9.2) and Africa (OR = 3.6, 95% CI = 1.7-7.7) and a diet without pork (OR = 0.5, 95% CI = 0.3-0.8 for pork eaters). CONCLUSIONS: E. coli host factors previously associated with higher pathogenicity were all more common in BSIs compared with carriers. This indicates that the risk of invasive infection with EPE may be relatively modest in many community carriers and that EPE carriage of high-risk strains should be the focus of attention for prevention.


Asunto(s)
Bacteriemia/epidemiología , Portador Sano/epidemiología , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Escherichia coli/epidemiología , Escherichia coli/genética , beta-Lactamasas/genética , Adolescente , Adulto , Anciano , Antibacterianos/uso terapéutico , Bacteriemia/diagnóstico , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Portador Sano/microbiología , Portador Sano/transmisión , Estudios Transversales , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/transmisión , Heces/microbiología , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Encuestas y Cuestionarios , Suecia/epidemiología , Adulto Joven
3.
Emerg Infect Dis ; 22(4): 634-40, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26982890

RESUMEN

Extended-spectrum ß-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.


Asunto(s)
Animales Domésticos/microbiología , Bacteriemia/epidemiología , Proteínas Bacterianas/genética , Infecciones por Escherichia coli/epidemiología , Escherichia coli/genética , Carne/microbiología , Enfermedades de las Aves de Corral/epidemiología , beta-Lactamasas/genética , Animales , Bacteriemia/microbiología , Bacteriemia/transmisión , Proteínas Bacterianas/metabolismo , Bovinos , Pollos/microbiología , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/transmisión , Microbiología de Alimentos , Expresión Génica , Humanos , Plásmidos/química , Plásmidos/metabolismo , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/transmisión , Suecia/epidemiología , Porcinos/microbiología , beta-Lactamasas/metabolismo
4.
Int J Food Microbiol ; 171: 8-14, 2014 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-24296257

RESUMEN

The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans and animals world-wide. Their occurrence in food-producing animals suggests that meat is a possible link between the two populations. This study investigated the occurrence and characteristics of Salmonella and ESBL- or pAmpC-producing E. coli in 430 samples of beef, pork and broiler meat imported into Sweden, in order to provide data required for assessing the potential public health risk of these bacteria in food. Depending on region of origin, ESBL/pAmpC-producing E. coli were found in 0-8% of beef samples, 2-13% of pork samples and 15-95% of broiler meat samples. The highest prevalence was in South American broiler meat (95%), followed by broiler meat from Europe (excluding Denmark) (61%) and from Denmark (15%). Isolates from meat outside Scandinavia were generally defined as multiresistant. A majority of the ESBL/pAmpC genes were transferable by conjugation. Bla(CTX-M-2) and bla(CTX-M-8) were the dominant genes in E. coli from South American broiler meat, whereas bla(CMY-2) and bla(CTX-M-1) dominated in European meat. The majority of bla(CMY-2) and bla(CTX-M-1) were situated on plasmids of replicon type incK and incI1, respectively. The same combinations of ESBL/pAmpC genes and plasmids have been described previously in clinical human isolates. Salmonella was found in five samples tested, from European pork and broiler meat. No Salmonella isolate was resistant to third-generation cephalosporins. In conclusion, meat imported into Sweden, broiler meat in particular, is a potential source of human exposure to ESBL- and pAmpC-producing E. coli.


Asunto(s)
Escherichia coli/fisiología , Microbiología de Alimentos , Carne/microbiología , Salmonella/fisiología , Animales , Antibacterianos/farmacología , Bovinos , Cefalosporinas/farmacología , Pollos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Europa (Continente) , Plásmidos/genética , Salmonella/efectos de los fármacos , Salmonella/enzimología , Salmonella/genética , Salmonella/aislamiento & purificación , América del Sur , Suecia , Porcinos
5.
Acta Vet Scand ; 55: 3, 2013 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-23336334

RESUMEN

BACKGROUND: The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the blaCTX-M-1, to determine if the spread was due to a specific clone. FINDINGS: Ten isolates carrying blaCTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The blaCTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole. CONCLUSION: The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying blaCTX-M-1.


Asunto(s)
Pollos , Infecciones por Escherichia coli/veterinaria , Escherichia coli/enzimología , Plásmidos/metabolismo , beta-Lactamasas/metabolismo , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Regulación Bacteriana de la Expresión Génica/fisiología , Plásmidos/genética , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/microbiología , Suecia/epidemiología , beta-Lactamasas/genética
6.
Appl Environ Microbiol ; 79(7): 2463-6, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23354705

RESUMEN

Forty-four percent of Swedish chicken meat fillets were contaminated with extended-spectrum or transferable AmpC beta-lactamase-producing Escherichia coli strains. Isolates from Swedish chicken meat and broilers were closely related to isolates from chicken meat imported into Sweden; these results indicate a common source of the contamination.


Asunto(s)
Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Carne/microbiología , beta-Lactamasas/genética , Animales , Pollos/microbiología , Escherichia coli/genética , Prevalencia , Suecia
7.
BMC Vet Res ; 8: 9, 2012 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-22280482

RESUMEN

BACKGROUND: Within the genera Chlamydia, the development of refined diagnostic techniques has allowed the identification of four species that are capable of infecting pigs. The epidemiology, clinical, and zoonotic impacts of these species are however largely unknown. The study aimed to investigate the presence of Chlamydia spp. in the intestines of growing pigs and in conjunctival swabs from finisher pigs, and relate the findings to clinical signs. RESULTS: By histology, 20 of 48 pigs had intestinal lesions that may be consistent with chlamydial infection. By PCR, forty-six of the pigs were positive whereas two samples were inhibited. Sequencing of 19 DNA extracts identified these as Chlamydia suis. By immunohistochemistry, 32 of 44 samples were positive and a significant relationship was detected between macroscopically visible intestinal lesions and a high degree of infection. By real-time PCR, a significant difference was detected between pigs with and without conjunctivitis when a Ct value of 36 was employed but not when a Ct value of 38 was employed. CONCLUSIONS: Chlamydia suis was demonstrated in most samples and overall, no correlation to clinical signs was detected. However, a correlation was noted between samples with a high degree of infection and the presence of clinical signs. It is possible, that the intensive pig production systems studied might predispose for the transmission and maintenance of the infection thus increasing the infectious load and the risk for disease in the pig.


Asunto(s)
Infecciones por Chlamydia/veterinaria , Chlamydia/aislamiento & purificación , Enfermedades de los Porcinos/microbiología , Animales , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Conjuntiva/microbiología , ADN Bacteriano , Intestinos/microbiología , Reacción en Cadena de la Polimerasa/veterinaria , Porcinos , Enfermedades de los Porcinos/patología
8.
Microb Drug Resist ; 18(1): 88-93, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21749212

RESUMEN

This study was conducted to elucidate the accuracy of the current streptomycin epidemiological cut-off value (ECOFF) for Escherichia coli and Salmonella spp. A total of 236 Salmonella enterica and 208 E. coli isolates exhibiting MICs between 4 and 32 mg/L were selected from 12 countries. Isolates were investigated by polymerase chain reaction for aadA, strA, and strB streptomycin resistance genes. Out of 236 Salmonella isolates, 32 (13.5%) yielded amplicons for aadA (n = 23), strA (n = 9), and strB (n = 11). None of the 60 Salmonella isolates exhibiting MIC 4 mg/L harbored resistance genes. Of the Salmonella isolates exhibiting MICs 8 mg/L, 16 mg/L, and 32 mg/L, 1.6%, 15%, and 39%, respectively, tested positive for one or more genes. For most monitoring programs, the streptomycin ECOFF for Salmonella is wild type (WT) ≤32 or ≤16 mg/L. A cut-off value of WT ≤32 mg/L would have misclassified 13.5% of the strains as belonging to the WT population, since this proportion of strains harbored resistance genes and exhibited MICs ≤32 mg/L. Out of 208 E. coli strains, 80 (38.5%) tested positive for aadA (n = 69), strA (n = 18), and strB (n = 31). Of the E. coli isolates exhibiting MICs of 4 mg/L, 8 mg/L, 16 mg/L, and 32 mg/L, 3.6%, 17.6%, 53%, and 82.3%, respectively, harbored any of the three genes. Based on the European Committee on Antimicrobial Susceptibility Testing guidelines (ECOFF ≤16 mg/L), 25% of the E. coli strains presenting MIC ≤16 mg/L would have been incorrectly categorized as belonging to the WT population. The authors recommend an ECOFF value of WT ≤16 mg/L for Salmonella and WT ≤8 mg/L for E. coli.


Asunto(s)
Antibacterianos/farmacología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/genética , Salmonelosis Animal/tratamiento farmacológico , Salmonella/genética , Estreptomicina/farmacología , Animales , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Europa (Continente)/epidemiología , Genes Bacterianos , Ganado , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Aves de Corral , Guías de Práctica Clínica como Asunto , Salmonella/efectos de los fármacos , Salmonelosis Animal/epidemiología , Salmonelosis Animal/microbiología
9.
Am J Vet Res ; 72(6): 806-12, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21627527

RESUMEN

OBJECTIVE: To investigate shedding of chlamydiae from conjunctiva and genital tracts of cats without clinical signs of conjunctivitis or other infectious disease in relation to their titers of serum antibodies against chlamydiae and to serum amyloid A (SAA) and serum α(1)-acid glycoprotein (AGP) concentrations. ANIMALS: 62 healthy cats. PROCEDURES: Serum from each cat was analyzed for antibodies against chlamydiae and for SAA and AGP concentrations. Swab samples from the conjunctival sac and genital tract were analyzed with a real-time PCR assay for Chlamydiaceae. RESULTS: 4 of 8 of cats with high antibody titers (ie, 1,600) shed chlamydiae, but only from the conjunctiva. Chlamydiae could not be detected in samples from cats with lower antibody titers nor from any genital tract samples. In cats with antibody titers of 1,600, mean ± SD SAA concentration was significantly higher when chlamydiae were detected in conjunctival swab samples (3.9 ± 1.0 mg/L) than when no chlamydiae were detected (1.4 ± 1.0 mg/L). However, SAA concentration was greater than the limit for an acute-phase response in only one of those cats. There was no significant difference in serum AGP concentrations between cats with high titers that were or were not shedding chlamydiae. Nine of 30 (30%) cats (5 with and 4 without detectable serum antibodies against chlamydiae) that had been mated developed reproductive disorders. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically normal cats with high chlamydiae-specific antibody titers can shed and thus transmit chlamydiae. Venereal spread from cats without clinical signs of infection is likely not common.


Asunto(s)
Reacción de Fase Aguda , Derrame de Bacterias , Enfermedades de los Gatos/transmisión , Infecciones por Chlamydia/veterinaria , Chlamydia/aislamiento & purificación , Aborto Veterinario/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Enfermedades de los Gatos/sangre , Enfermedades de los Gatos/microbiología , Gatos , Infecciones por Chlamydia/sangre , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/transmisión , Conjuntiva/microbiología , Femenino , Masculino , Orosomucoide/análisis , Pene/microbiología , Reacción en Cadena de la Polimerasa , Proteína Amiloide A Sérica/análisis , Vagina/microbiología
10.
Acta Vet Scand ; 52: 7, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-20122147

RESUMEN

After 27 years with no detected cases, an outbreak of anthrax occurred in a beef cattle herd in the south of Sweden. The outbreak was unusual as it occurred in winter, in animals not exposed to meat-and-bone meal, in a non-endemic country. The affected herd consisted of 90 animals, including calves and young stock. The animals were kept in a barn on deep straw bedding and fed only roughage. Seven animals died during 10 days, with no typical previous clinical signs except fever. The carcasses were reportedly normal in appearance, particularly as regards rigor mortis, bleeding and coagulation of the blood. Subsequently, three more animals died and anthrax was suspected at necropsy and confirmed by culture and PCR on blood samples. The isolated strain was susceptible to tetracycline, ciprofloxacin and ampicillin. Subtyping by MLVA showed the strain to cluster with isolates in the A lineage of Bacillus anthracis. Environmental samples from the holding were all negative except for two soil samples taken from a spot where infected carcasses had been kept until they were picked up for transport. The most likely source of the infection was concluded to be contaminated roughage, although this could not be substantiated by laboratory analysis. The suspected feed was mixed with soil and dust and originated from fields where flooding occurred the previous year, followed by a dry summer with a very low water level in the river allowing for the harvesting on soil usually not exposed. In the early 1900s, animal carcasses are said to have been dumped in this river during anthrax outbreaks and it is most likely that some anthrax spores could remain in the area. The case indicates that untypical cases in non-endemic areas may be missed to a larger extent than previously thought. Field tests allowing a preliminary risk assessment of animal carcasses would be helpful for increased sensitivity of detection and prevention of further exposure to the causative agent.


Asunto(s)
Carbunco/veterinaria , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Animales , Carbunco/diagnóstico , Carbunco/epidemiología , Carbunco/microbiología , Antibacterianos/farmacología , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/genética , Bacillus anthracis/aislamiento & purificación , Bovinos , Enfermedades de los Bovinos/epidemiología , Pruebas de Sensibilidad Microbiana , Microbiología del Suelo , Bazo/microbiología , Suecia
11.
Acta Vet Scand ; 52: 2, 2010 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-20070900

RESUMEN

BACKGROUND: Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission. METHODS: Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier ELISA Cp. abortus serum verification kit. RESULTS: No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected. CONCLUSIONS: The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.


Asunto(s)
Enfermedades de los Bovinos/microbiología , Infecciones por Chlamydophila/veterinaria , Chlamydophila/aislamiento & purificación , Epidídimo/microbiología , Semen/microbiología , Crianza de Animales Domésticos , Animales , Anticuerpos Antibacterianos , Cruzamiento , Bovinos , Enfermedades de los Bovinos/transmisión , Chlamydophila/genética , Chlamydophila/inmunología , Infecciones por Chlamydophila/microbiología , Infecciones por Chlamydophila/transmisión , ADN Bacteriano , Femenino , Fertilidad , Masculino , Estudios Seroepidemiológicos
12.
Acta Vet Scand ; 50: 39, 2008 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-18822135

RESUMEN

BACKGROUND: Reports worldwide indicate high prevalence of Chlamydophila spp. infection in cattle. To assess the prevalence in Sweden, 525 cows in 70 dairy herds with reproductive disorders was investigated. METHODS: To detect antibodies two commercially available kits were used. Moreover, 107 specimens, including vaginal swabs, organ tissues and milk were analysed by Polymerase Chain Reaction (PCR). RESULTS: Two (0.4%) cows were seropositive in the Pourquier Cp. abortus ELISA. The seroprevalence with the Chekit ELISA was 28% with no difference between cases and controls. Five specimens were positive in real-time PCR and further analysed by nested PCR. Cp. pecorum was confirmed by partial omp1 DNA sequencing of the nested PCR product of vaginal swabs from control cows. CONCLUSION: The results suggest that Cp. abortus infection is absent or rare in Swedish cows whereas Cp. pecorum is probably more spread. They also suggest that Chlamydophila spp. are not related to reproduction disorders in Swedish cattle.


Asunto(s)
Aborto Veterinario/microbiología , Enfermedades de los Bovinos/microbiología , Infecciones por Chlamydophila/veterinaria , Chlamydophila/aislamiento & purificación , Aborto Veterinario/epidemiología , Animales , Anticuerpos Antibacterianos/sangre , Bovinos , Enfermedades de los Bovinos/epidemiología , Chlamydophila/genética , Infecciones por Chlamydophila/epidemiología , Infecciones por Chlamydophila/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Reacción en Cadena de la Polimerasa/veterinaria , Estudios Seroepidemiológicos , Suecia/epidemiología
13.
Vet Microbiol ; 96(3): 277-87, 2003 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-14559175

RESUMEN

There is an increasing demand for fast and reliable methods to distinguish Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from closely related mycobacteria and also a need for rapid strain specific typing of clinical isolates for epidemiological reasons. In the present study, the potential of rep-PCR as a fingerprinting method for M. paratuberculosis was assessed and compared to conventional RFLP. A PCR assay was designed and optimised to obtain reproducible fingerprints of mycobacterial DNA with primers targeting the enterobacterial intergenic consensus (ERIC) sequence and the M. paratuberculosis specific insertion sequence IS900. Reproducible fingerprints were obtained with 60 strains of M. paratuberculosis, 16 strains of M. avium subsp. avium, 3 strains of M. intracellulare, and 11 other mycobacterial strains. A species-specific band pattern that was clearly distinguishable from that of other mycobacteria was obtained with M. paratuberculosis. The rep-PCR did not detect any differences among M. paratuberculosis strains of different RFLP types, and was therefore not considered as an alternative fingerprinting method. However, the species-specific band pattern make IS900/ERIC-PCR a suitable alternative for distinguishing M. paratuberculosis from other mycobacteria, especially in cases of IS900 PCR positive mycobacteria. The fingerprinting method reported was fast and easy to perform, and produced highly reproducible results.


Asunto(s)
Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculosis/diagnóstico , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Southern Blotting/veterinaria , Dermatoglifia del ADN/métodos , Dermatoglifia del ADN/veterinaria , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Mycobacterium avium subsp. paratuberculosis/clasificación , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los Resultados , Especificidad de la Especie
14.
J Vet Diagn Invest ; 15(3): 268-73, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12735349

RESUMEN

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors werepresent. When fecal samples were spiked with the mimic, the detection limit was 10(2) molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 10(3) mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.


Asunto(s)
Heces/microbiología , Lawsonia (Bacteria)/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de los Porcinos/diagnóstico , Animales , Artefactos , Clonación Molecular , Lawsonia (Bacteria)/genética , Imitación Molecular/genética , Estándares de Referencia , Porcinos/microbiología , Enfermedades de los Porcinos/microbiología
16.
Vet Microbiol ; 87(4): 327-40, 2002 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-12069770

RESUMEN

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.


Asunto(s)
Enfermedades de los Bovinos/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Bovinos , Centrifugación por Gradiente de Densidad/veterinaria , Recuento de Colonia Microbiana/veterinaria , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Heces/microbiología , Mycobacterium avium subsp. paratuberculosis/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Povidona , Sensibilidad y Especificidad , Dióxido de Silicio , Suecia
17.
FEMS Microbiol Lett ; 209(2): 267-71, 2002 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-12007816

RESUMEN

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.


Asunto(s)
Elementos Transponibles de ADN/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium/genética , Animales , Bovinos , Mycobacterium avium/clasificación , Mycobacterium avium subsp. paratuberculosis/clasificación , Fenotipo , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética
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