An evaluation of the ability of Dichelobacter nodosus to survive in soil

Background

Dichelobacter nodosus is the causative agent of footrot in sheep. The survival of the bacterium in soil is of importance for the epidemiology of the disease. The investigation evaluates the survival of D. nodosus in soil with and without added hoof powder stored under different temperatures.

Results

An experimental setup was used with bacteriological culture and real-time polymerase chain reaction (PCR), and the results indicate that the bacteria can survive in soil for longer time than previously expected. The survival time was found to be dependent on temperature and the addition of hoof powder to the soil, with the longest survival time estimated to be 24 days in soil samples with hoof powder stored at 5°C.

Conclusion

Our findings indicate that the survival time of D. nodosus and its ability to infect susceptible sheep on pasture under different climatic conditions should be studied further.     

Prevalence of footrot in Swedish slaughter lambs

Background

Footrot is a world-wide contagious disease in sheep and goats. It is an infection of the epidermis of the interdigital skin, and the germinal layers of the horn tissue of the feet. The first case of footrot in Swedish sheep was diagnosed in 2004. Due to difficulties in distinguishing benign footrot from early cases of virulent footrot and because there is no possibility for virulence testing of strains of Dichelobacter nodosus in Sweden, the diagnosis is based of the presence or absence of clinical signs of footrot in sheep flocks. Ever since the first diagnosed case the Swedish Animal Health Service has worked intensively to stop the spread of infection and control the disease at flock level. However, to continue this work effectively it is important to have knowledge about the distribution of the disease both nationally and regionally. Therefore, the aims of this study were to estimate the prevalence of footrot in Swedish lambs at abattoirs and to assess the geographical distribution of the disease.

Methods

A prevalence study on footrot in Swedish lambs was performed by visual examination of 2000 feet from 500 lambs submitted from six slaughter houses. Each foot was scored according to a 0 to 5 scoring system, where feet with score ≥2 were defined as having footrot. Moreover, samples from feet with footrot were examined for Dichelobacter nodosus by culture and PCR.

Results

The prevalence of footrot at the individual sheep level was 5.8%, and Dichelobacter nodosus was found by culture and PCR in 83% and 97% of the samples from feet with footrot, respectively. Some minor differences in geographical distribution of footrot were found in this study.

Conclusions

In a national context, the findings indicate that footrot is fairly common in Swedish slaughter lambs, and should be regarded seriously.     

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen <Emphasis Type="Italic">Dichelobacter nodosus</Emphasis>

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.     

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway

Background

In 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination.

Results

The Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant.

Conclusion

This study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.     

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log10⁵ to log10⁷Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 10⁴–10⁷ mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.     

Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

Background

Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs.

Methods

Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations.

Results

The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs.

Conclusion

Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow.     

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping).The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.     

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

Background

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.

Methods

Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates.

Results

Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05).

Conclusions

The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.     

Clear distinction between Burkholderia mallei and Burkholderia pseudomallei using fluorescent motB primers

Background

A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers.

Findings

Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively.

Conclusions

This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13028-015-0104-4) contains supplementary material, which is available to authorized users.     

Investigation of Chlamydophila spp. in dairy cows with reproductive disorders

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.     

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

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.     

Molecular analysis of Dichelobacter nodosus isolated from footrot in sheep in Malaysia

Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5′GGGCCC3′), SfiI (5′GGCCNNNNNGGCC3′)and SmaI (′5CCCGGG3′) enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.     

A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids

Background

Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated.

Findings

A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested.

Conclusions

The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field.     

A longitudinal study of Campylobacter distribution in a turkey production chain

Background

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis.Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.

Methods

Samples (N = 456) were regularly collected from one turkey parent flock, the hatchery, six different commercial turkey farms and from 11 different stages at the slaughterhouse. For the detection of Campylobacter, a conventional culture and a PCR method were used. Campylobacter isolates (n = 143) were identified to species level by a multiplex PCR assay.

Results

No Campylobacter were detected in either the samples from the turkey parent flock or from hatchery samples using the culture method. PCR detected Campylobacter DNA in five faecal samples and one fluff and eggshell sample. Six flocks out of 12 commercial turkey flocks where found negative at the farm level but only two were negative at the slaughterhouse.

Conclusion

During the brooding period Campylobacter might have contact with the birds without spreading of the contamination within the flock. Contamination of working surfaces and equipment during slaughter of a Campylobacter positive turkey flock can persist and lead to possible contamination of negative flocks even after the end of the day''s cleaning and desinfection. Reduction of contamination at farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of contaminated poultry meat in Finland. Due to the low numbers of Campylobacter in the Finnish turkey production chain, enrichment PCR seems to be the optimal detection method here.     

Fusobacterium necrophorum,and not Dichelobacter nodosus,is associated with equine hoof thrush

The aim of this study was to determine which of the two species, Fusobacterium necrophorum or Dichelobacter nodosus, are associated with hoof thrush in horses. Fourteen hoof samples, collected from eight horses with thrush and 14 samples collected from eight horses with healthy hooves, were examined for the presence of F. necrophorum, Fusobacterium equinum and D. nodosus. Only isolates with phenotypic characteristics representing Fusobacterium could be cultured. Total DNA extracted from the 28 hoof samples was amplified by using DNA primers designed from gene lktA, present in F. necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. equinum, and gene fimA, present in D. nodosus. The lktA gene was amplified from five of the 14 infected hoof samples and from one hoof sample without thrush. The DNA sequence of the amplified ltkA gene was identical to the lktA gene of the type strain of F. necrophorum (GenBank accession number AF312861). The isolates were phenotypically differentiated from F. equinum. No DNA was amplified using the fimA primer set, suggesting that F. necrophorum, and not D. nodosus, is associated with equine hoof thrush. Hoof thrush in horses is thus caused by F. necrophorum in the absence D. nodosus. This is different from footrot in sheep, goats, cattle and pigs, which is caused by the synergistic action of F. necrophorum and D. nodosus.     

Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds

Background

Bovine babesiosis is regarded as a limited health problem for Norwegian cows, and the incidence has decreased markedly since the 1930s. Rare cases of babesiosis in splenectomised humans from infection with Babesia divergens and B.venatorum have been described. The objective of this study was to determine whether birds can introduce Babesia-infected ticks. There are between 30 and 85 million passerine birds that migrate to Norway every spring.

Methods

Passerine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses.

Results

Of 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001).

Conclusions

Birds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.     

Abattoir-based study on the epidemiology of caprine tuberculosis in Ethiopia using conventional and molecular tools

Background

Despite the important role of goats for meat and milk production in Ethiopia, little information is available on the epidemiology of caprine tuberculosis (TB). Caprine TB is important as milk is usually consumed raw particularly by Ethiopian pastoralists. The objectives of the present study were to estimate the prevalence of TB in goats at an abattoir, to evaluate associated risk factors and to characterize the causative mycobacteria.

Methods

A cross-sectional study was conducted on 1990 randomly selected male goats that were slaughtered at Luna Export Abattoir of central Ethiopia. Postmortem examination, mycobacterial culturing and molecular typing techniques like genus typing, deletion typing and spoligotyping were used.

Result

The overall prevalence of caprine TB-like lesions was 3.5%. The lesion prevalence increased significantly with increasing age. Mycobacteria were found by culture and seen as acid fast bacilli in 12% of the goats with TB-like lesions. Characterization of the eight isolates using multiplex polymerase chain reaction (PCR) indicated that five of them belonged to the genus Mycobacterium. Four of the latter were confirmed to be members of the M. tuberculosis complex. Further characterization of the three M. tuberculosis isolates by spoligotyping identified them as type SIT53 and two new spoligotypes.

Conclusion

The isolation of M. tuberculosis from goats in this study indicates a potential risk of transmission of M. tuberculosis between humans and goats.