Stina Englund

Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johnes disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.

Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden

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.

The use of a mimic to detect polymerase chain reaction- inhibitory factors in feces examined for the presence of Lawsonia intracellularis

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 were present. When fecal samples were spiked with the mimic, the detection limit was 102 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 103 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.

Anthrax outbreak in a Swedish beef cattle herd - 1st case in 27 years: Case report

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.

Frequent Occurrence of Extended-Spectrum Beta-Lactamase- and Transferable AmpC Beta-Lactamase-Producing Escherichia coli on Domestic Chicken Meat in Sweden

ABSTRACT 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.

Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1

BackgroundThe 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.FindingsTen 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.ConclusionThe 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.

Investigation of Chlamydophila spp. in dairy cows with reproductive disorders

BackgroundReports 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.MethodsTo 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).ResultsTwo (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.ConclusionThe 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.

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

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.

Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

BackgroundReproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.MethodsSemen 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.ResultsNo chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.ConclusionsThe 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.

Limited Dissemination of Extended-Spectrum β-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

Food is a limited source of these antimicrobial resistance genes for humans.