Botulism in Cattle: A Case Report of an Outbreak in Sardinia (Italy).

Clostridium botulinum is the main causative agent of botulism in humans and animals. The ingestion of the botulinum neurotoxin, usually types C and D, has been shown to produce disease (neurological symptoms) in most botulism cases in cattle. We report an outbreak in Southern Sardinia that involved a livestock farm with 120 animals, 39 of which died. The aim of this report is to describe the course of this outbreak and the progression of symptoms up to the death of some animals; we also describe the therapeutic approach applied in this case and the analytical techniques used to diagnose the disease. Finally, we emphasize the importance of promptly proceeding with the sampling of several matrixes when a suspicion of botulism arises.

Infant Botulism: Checklist for Timely Clinical Diagnosis and New Possible Risk Factors Originated from a Case Report and Literature Review.

Infant botulism is a rare and underdiagnosed disease caused by BoNT-producing clostridia that can temporarily colonize the intestinal lumen of infants less than one year of age. The diagnosis may be challenging because of its rareness, especially in patients showing atypical presentations or concomitant coinfections. In this paper, we report the first infant botulism case associated with Cytomegalovirus coinfection and transient hypogammaglobulinemia and discuss the meaning of these associations in terms of risk factors. Intending to help physicians perform the diagnosis, we also propose a practical clinical and diagnostic criteria checklist based on the revision of the literature.

The First Case of Botulism in a Donkey.

Botulism, a severe neuroparalytic disease that can affect humans, all warm-blooded animals, and some fishes, is caused by exotoxins produced by ubiquitous, obligate anaerobic, spore-forming bacteria belonging to the genus Clostridium and named botulinum neurotoxin (BoNT)-producing clostridia. This report presents the case of a 3-year-old donkey mare referred for progressive and worsening dysphagia of four days' duration. Her voluntary effort in eating and drinking was conserved, and she was able to slow chew without swallowing. A complete neurological examination was performed, and botulism was strongly suspected. The ability to swallow feed and water returned on the tenth day of hospitalization and improved progressively. The jenny was discharged from the hospital after fifteen days. During the hospitalization, the Italian National Reference Centre for Botulism confirmed the diagnosis: mare's feces were positive for BoNT/B and Clostridium botulinum type B.

Foodborne botulism: an evolving public health challenge.

Foodborne botulism is a life-threatening disease caused by the ingestion of food containing preformed botulinum neurotoxins, the most potent natural poisons known to humans. On the basis of the new challenges in management of the diseases as well as considering the potential use of botulinum toxins as biological weapons, foodborne botulism is still considered a public health emergency. Each suspected case should be immediately notified to public health authorities with the aim of preparing a prompt response. With the aim of improving botulism surveillance systems, health authorities as well as governmental organizations should enhance national and international cooperation. Education and training activities devoted to operators involved in the disease management, and to general population, may significantly contribute to strengthen the system.

Galleria mellonella as an in vivo model for assessing the protective activity of probiotics against gastrointestinal bacterial pathogens.

The antagonistic activity against gastrointestinal bacterial pathogens is an important property of probiotic bacteria and a desirable feature for pre-selection of novel strains with probiotic potential. Pre-screening of candidate probiotics for antibacterial activity should be based on in vitro and in vivo tests. This study investigated whether the protective activity of probiotic bacteria against gastrointestinal bacterial pathogens can be evaluated using Galleria mellonella larvae as an in vivo model. Larvae were pre-inoculated with either of two widely used probiotic bacteria, Lactobacillus rhamnosus GG or Clostridium butyricum Miyairi 588, and then challenged with Salmonella enterica Typhimurium, enteropathogenic Escherichia coli or Listeria monocytogenes. Survival rates increased in the probiotic pretreated larvae compared with control larvae inoculated with pathogens only. The hemocyte density increased as well in the probiotic pretreated larvae, indicating that both probiotics induce an immune response in the larvae. The antibacterial activity of probiotics against the pathogens was also assayed by an in vitro agar spot test: results were partially consistent with those obtained by the G. mellonella protection assay. The results obtained, as a whole, suggest that G. mellonella larvae are a potentially useful in vivo model that can complement in vitro assays for pre-screening of candidate probiotics.

Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E.

Structure and genetic content of the megaplasmids of neurotoxigenic clostridium butyricum type E strains from Italy.

We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.

Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains.

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.

National survey outcomes on commercial probiotic food supplements in Italy.

To assess whether the probiotic food supplements, produced and distributed on the Italian market during 2005-2006, complied with the Italian Guidelines on Prebiotics and Probiotics, 72 samples from 29 processing plants were analyzed. The survey included 41 samples from processing plants and 31 samples of the same brand from retailers collected at timed intervals (3, 8 and 13 months). A polyphasic approach based on a suitable analytical collection method (genotypic identification of total bacteria - differential presumptive enumeration - genotypic identification of viable bacteria) was adopted to identify and quantify the microorganisms labelled and recovered from the probiotic supplements examined. Most supplements analyzed (87%) did not conform to the Italian guidelines and the differences were both quantitative and qualitative (number determination, purity, types and viability of microorganisms). Even though most labelled supplements (25 samples) indicated the presence of Bifidobacterium bifidum, this organism was only detected sporadically and always as dead cells. Unexpected results were obtained during our survey due to the absence of viability of Bacillus coagulans spores in some labelled supplements. Besides this, some of these supplements also contained other spore-forming species, identified as B. cereus that are toxin producing. We have also documented a widespread use of misclassified microbial species or species with fictitious names. The main factors involved in the absence of compliance were examined and the poor quality control applied by manufacturers was emphasized.

Cervicofacial emphysema secondary to facebow injury: a case report.

AIM: To report the clinical case of a child with facial and periorbital emphysema caused by an orthodontic device. CASE REPORT: An 11-year-old child presented to our clinic showing moderate swelling of the left facial area. Based on his dental history, physical findings, and instrument examinations, the diagnosis of cervicofacial emphysema was established, caused by disengagement of the facebow. One week later, all swelling and crepitus had disappeared without complications. Most patients who develop subcutaneous emphysema after a dental procedure have only moderate local swelling, which normally resolves spontaneously and without complications within a week. However the spread of large amounts of air into the deeper spaces may cause life-threatening sequelae. CONCLUSIONS: Orthodontists should be aware that the use of extraoral traction applied via a facebow can cause soft tissue injures and emphysema of the cervicofacial region. It is important to avoid misdiagnosis and to appropriately inform patient and parents about this condition.

Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains.

BACKGROUND: Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont/B subtype-related. METHODOLOGY/PRINCIPAL FINDINGS: PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from approximately 55 kb to approximately 245 kb. CONCLUSIONS/SIGNIFICANCE: The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.

The survival of hepatitis A virus in fresh produce.

Fresh produce has been repeatedly implicated as the source of human viral infections, including infection with hepatitis A virus (HAV). The objective of the present study was to evaluate the HAV adsorption capacity of the surface of various fresh vegetables that are generally eaten raw and the persistence of the HAV. To this end, the authors experimentally contaminated samples of lettuce, fennel, and carrot by immersing them in sterile distilled water supplemented with an HAV suspension until reaching a concentration of 5 log tissue culture infectious dose (TCID50)/ml. After contamination, the samples were stored at 4 degrees C and analysed at 0, 2, 4, 7, and 9 days. To detect the HAV, RT-nested-PCR was used; positive samples were subjected to the quantitative determination using cell cultures. The three vegetables differed in terms of their adsorption capacity. The highest quantity of virus was consistently detected for lettuce, for which only a slight decrease was observed over time (HAV titre = 4.44 +/- 0.22 log TCID50/ml at day 0 vs. 2.46 +/- 0.17 log TCID50/ml at day 9, before washing). The virus remained vital through the last day of storage. For the other two vegetables, a greater decrease was observed, and complete inactivation had occurred at day 4 for carrot and at day 7 for fennel. For all three vegetables, washing does not guarantee a substantial reduction in the viral contamination.