Ilenia Drigo

Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

ABSTRACT Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C (n = 12), C-D (n = 29), D (n = 5), and D-C (n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains (n = 20), non-BoNT-producing clostridia (n = 20), and other bacterial species (n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n = 108), poultry (n = 60), and bovines (n = 56). Among the 292 samples, 144 were positive for either the bont/C-D or the bont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

First reporting of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in an industrial rabbit holding and in farm-related people.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been described in food-producing animals and farm or slaughterhouse workers involved in the primary industrial production of swine, bovine and poultry. This communication describes the first case of LA-MRSA (ST398, spa types t034 and t5210) occurring in rabbits raised intensively for meat production and involving farm workers or their family members. In 2012-2013, in a study involving 40 rabbit industrial holdings in Italy, one farm was found to have rabbits colonized or infected with MRSA. Four farm workers and one of their relatives were found to be carrying MRSA. In this case holding, rabbits, people and the holding environment were further investigated and followed up by a second sampling five months later. MRSA was found in 48% (11/23) and 25% (15/59) of the rabbits carrying S. aureus at first and second samplings, respectively. Five months after first detection, some farm workers or family members were still MRSA carriers. Surface samples (2/10) and air samples (2/3) were contaminated with MRSA. Air samples yielded MRSA counts of 5 and 15CFU/m(3). MRSA from rabbits and people collected at first sampling were spa types t034 and t5210 belonging to ST398. The MRSA isolates from rabbits and persons tested at second sampling were t034 and t5210, but spa types t1190 and t2970 were also detected in MRSA isolates from rabbits. Tracing the epidemiological pattern earlier may prevent further spread of LA-MRSA in these food producing animals.

A survey of Clostridium spiroforme antimicrobial susceptibility in rabbit breeding

Rabbit meat breeding may be heavily affected by enterotoxaemia due to Clostridium spiroforme. Data on its antimicrobial susceptibility are insufficient, presumably because of difficulties in cultivating and identifying the pathogen. Our aim is therefore to provide this information to veterinary practitioners by focusing on a panel of therapeutics used in intensive rabbit units. Lincomycin was also checked in order to investigate the origin of resistance to macrolides. Minimal inhibitory concentrations (MICs) were determined with the agar dilution method according to the CLSI M11-A7 protocol (2007). MIC(50) and MIC(90) were, respectively, 64 and 64microg/ml for tiamulin, 32 and 32microg/ml for norfloxacin, 0.063 and 0.125microg/ml for amoxicillin, and 8 and 16microg/ml for doxycycline. MIC(50) and MIC(90) were 256microg/ml for sulphadimethoxine, spiramycin and lincomycin. Our results have shown that intrinsic or acquired antimicrobial resistances are diffuse in the C. spiroforme population and suggest focusing on prevention rather than on treatment of clostridial overgrowth, by reducing risk factors and using antimicrobials prudently.

Toxin genotyping of Clostridium perfringens field strains isolated from healthy and diseased chickens

Clostridium perfringens is well known as the aetiological agent of necrotic enteritis in chicken. Type A and type c are considered the C. perfringens toxin types responsible for this disease. The aim of this study was to determine the presence of genes coding for α, β, ε, ι, β2 and enterotoxin in C. perfringens field strains collected from healthy and diseased chickens. Thirty-seven C. perfringens field strains were toxin typed: all strains resulted to be toxin type A and 3 of these tested positive for the presence of the toxin β2 coding gene. Four isolates showed the cpa gene with the insertion of a group II intron. Our findings confirm the most recent results reported from different countries and the data suggest that the role of C. perfringens type c should be revaluated in the etiopathogenesis of necrotic enteritis.

Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instruments default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instruments ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred. Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain. An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study. Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds. Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off. We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Molecular characterization and antimicrobial susceptibility of Clostridium difficile isolated from rabbits raised for meat production

Clostridium difficile is an important cause of enteric disease in humans and animals. Recent studies demonstrated a genetic overlap between C. difficile isolated from animals and humans suggesting animals as possible reservoir for human pathogenic strains. This study was a preliminary investigation on the occurrence of C. difficile in rabbits raised in industrial holdings for food production and aimed to characterise isolates and estimate their antimicrobial susceptibility. C. difficile isolates were characterized by toxin profiles, toxinotyping and PCR-ribotyping. The MICs of six antibiotics were determined using E-test. Between 2007 and 2013, 285 industrial holdings (representing 40% of the national census) submitted rabbits to our laboratory for diagnostic purposes, among these holdings, groups of three to five post-weaned rabbits were sampled once by convenience. 1279 samples of caecal content were collected. The overall isolation rate of C. difficile from the enteric specimen was 3% (38/1279), with no difference among animals affected or not by enteric disorders. Among isolates 66% (25/38) were toxigenic. Sixteen different PCR-ribotypes (RTs) were identified. Among the toxigenic strains RT-014/020, RT-078 and RT-012 were found in at least three rabbit holdings. According to the ECOFF threshold, 82% (31/38) C. difficile isolates displayed a reduced susceptibility to at least one and 18% (7/38) to three tested antimicrobials. Rabbits are colonized by heterogeneous C. difficile ribotypes many of which are commonly isolated in humans. One third of isolates displayed a reduced susceptibility to MTZ, the first choice antimicrobial for human CDI treatment. According to our findings rabbits are a potential source of C. difficile for humans.

Application of a PCR method for the diagnosis of ulcerative enteritis: preliminary results.

Abstract Ulcerative enteritis or “quail disease” is an acute clostridial infection of young birds reported in many avian species, chicken and turkey included. Clostridium colinum is the causative agent of ulcerative enteritis and because of the difficulties bound to the isolation and identification of this bacterium by means of classic bacteriological techniques, its detection appears very hard and the prevalence of this disease could be underestimated. To investigate the diffusion of C. colinum in enteric disease of birds, a recently developed PCR protocol was applied to 42 cultural broths previously inoculated with organs and intestinal samples collected from diseased subjects. PCR-positive broths were cultivated to attempt the isolation of C. colinum. Samples collected from positive birds were subjected to histological examinations. 4 birds (3 broilers chickens and 1 pigeon) resulted PCR-positive and, in one case, C. colinum was isolated. Gross and histological lesions of positive birds were compatible with those described in other ulcerative enteritis outbreaks. These preliminary results demonstrates that C. colinum is sporadically implicated in enteric diseases of broiler chickens (14.2%). In addition, the PCR assay proved to be an useful and reliable instrument to support the diagnosis of ulcerative enteritis and to facilitate the isolation of C. colinum.

Diffusion of Clostridium perfringens NetB positive strains in healthy and diseased chickens

Abstract For over 30 years α toxin was considered the key virulence factor responsible for the appearance of necrotic enteritis (NE) in chickens but, recently, a new toxin related to the occurrence of NE, called NetB, has been described. The aim of this work was to evaluate the CP toxin-type and the NetB gene presence in strains collected from chickens affected or not by enteric diseases. 107 strains were tested: 30 isolated from chickens affected by NE, 54 from subjects affected by other enteric pathologies and 22 from healthy animals. All strains resulted toxin-type A and 26.17% of these was positive also for β2 toxin gene. No strains were positive for cpe gene. 27% (29/107) of CP was NetB positive and 93% (27/29) of these was isolated from birds affected by intestinal disorders. 16 NetB positive strains were obtained from chickens affected by NE (16/30), 9 from animals affected by other intestinal disorders (9/54) and 4 from healthy animals (4/22). A significant difference between the number of NetB positive strains isolated from animals affected by NE and healthy chickens has been observed (P=0.014). However, the finding that the 17.4% of strains isolated from healthy chickens was also positive for NetB, confirm that other virulence factors could play an important role on NE appearance.

Longitudinal study on antimicrobial consumption and resistance in rabbit farming

Reliable indicators of antimicrobial consumption (AMC) measured with harmonised data and supported by indicators for antimicrobial resistance (AMR) at herd level are necessary to target antimicrobial misuse in food-producing animals. AMC data in 2010-2015 in 32 Italian industrial rabbit holdings weighted with semester production and standardised with animal daily doses (ADDs) were collected. Herd-level AMR against eight antimicrobials was assessed in Escherichia coli, Enterococcus faecalis and Enterococcus hirae collected in 2014-2015. Escherichia coli were assessed for mcr-1 and mcr-2 genes. To produce 1 kg of live rabbit, a mean of 71.8 ADDs was used. Overall AMC reduced over time (P < 0.05) owing to lowering consumption of tetracyclines (P < 0.05) and colistin (P < 0.01), but consumption of quinolones (P < 0.05), bacitracin (P < 0.01) and sulfonamides (P = 0.017) increased. All except one indicator E. coli were wild-type for cefotaxime, whereas 97% displayed reduced susceptibility to tetracyclines, 89% to trimethoprim, 63% to enrofloxacin, 24% to chloramphenicol and 21% to colistin. mcr-1 was detected in 50/320 E. coli isolates from 15/32 holdings; mcr-2 was not detected in 58 isolates with colistin MIC ≥ 2 mg/L. All 305 enterococci were wild-type for ampicillin, ciprofloxacin and vancomycin and displayed reduced tetracycline susceptibility. The mean antimicrobial resistance index (ARI) was 0.5 for E. coli and 0.3 for enterococci. ARI was significantly correlated with AMC at herd level for enterococci (P = 0.008) but not E. coli where high ARI levels were found in a few holdings with low AMC.