Botulinum Type Research Articles

An outbreak of botulism on a pig farm due to the newly described Clostridium botulinum type C

This study reports a botulism outbreak on a pig farm. Clostridium botulinum type C was detected using PCR. The gene encoding the toxin corresponds to a novel type C neurotoxin recently described in a human botulism outbreak, raising the question of its prevalence in pigs and the related risks to humans.

Functional prediction and assignment of Clostridium botulinum type A1 operome: A quest for prioritizing drug targets

Clostridium botulinum strain Hall produces potent botulinum neurotoxin type A1, which causes food-borne, infant, and wound botulism in humans. Antibiotics and botulinum antitoxins can control growth and prevent botulinum toxicity. However, limited information on a protein with an unknown function hinders the discovery of new drug targets for this disease. In this study, a combined bioinformatics approach with literature support was applied to predict, assign, and validate operome functions. Our functional annotation scheme was based on sequence motifs, conserved domains, structures, protein folds, and evolutionary relationships. Approximately 14.62 % of the operome exhibited sequence similarity to known proteins, with 6.65 % predicted functions for 293 proteins, including 121 proteins exclusive to C. botulinum. Structural analysis revealed a significant presence of the Rossmann fold (26 %) and miscellaneous folds (43 %) among the operome. Transporters (>85) and transcriptional regulators (>45) were prevalent, underscoring their importance in C. botulinum adaptive strategies. The newly identified operome contributed to the diverse cellular and metabolic processes of this organism. The function of its operome was involved in amino acid metabolism and botulinum neurotoxin biosynthesis. In this study, we identified and characterized 13 new virulence proteins from the operome to determine their structure–function relationships. These new metabolic and virulence proteins allow the organism to colonize and interact with the human gastrointestinal tract. This study provides a quest for new drugs and targets for treating the underlying diseases of C. botulinum in humans.

Botulism in fish: a review.

Published information about fish botulism is scant. We review here the current literature on fish botulism. Freshwater fish are susceptible to botulism. Only anecdotal evidence exists about possible botulism cases in saltwater fish. With only a few exceptions, the etiology of all cases of fish botulism reported is Clostridium botulinum type E, although fish are sensitive to, and may carry, various C. botulinum types. Clinical signs of botulism in fish include loss of equilibrium and motion, abducted opercula, open mouths, dark pigmentation, and head up/tail down orientation in which attempts to swim result in breaching the surface of the water. Dark pigmentation is thought to be associated with acetylcholine imbalance in botulinum neurotoxin (BoNT)-affected fish. Rarely, but similar to the situation in other animal species, fish can recover from botulism. Fish botulism can cause secondary outbreaks of the disease in birds, as botulism-affected fish stand out from normal fish, and are selectively preyed upon by fish-eating birds, which thus become intoxicated by the BoNT present in sick fish. The source of BoNT in fish has not been definitively confirmed. Fish may ingest C. botulinum spores that then germinate in their digestive tract, but the possibility that fish ingest preformed BoNT from the environment (e.g., dead fish, shellfish, insects) cannot be ruled out. The presumptive diagnosis of botulism in fish is established based on clinical signs, and as in other species, confirmation should be based on detection of BoNT in intestinal content, liver, and/or serum of affected fish.

Closed genome sequence of Clostridium botulinum type B1 strain isolated from an infant botulism case in the United States.

We present the closed genome sequence of the Clostridium botulinum BT-22100019 strain isolated from the stool specimen of an infant diagnosed with botulism. With 4.33-Mb genome size and 28.0% G + C content, the bont/B1 gene encoded for botulinum neurotoxin serotype B was found on a 262 kb plasmid arranged in a ha+ orfx - cluster.

Complete genomes of Clostridium botulinum type B(F) isolates associated with a 1995 foodborne botulism outbreak from commercial pâté reveals a recombination event disrupting the ntnh gene.

Foodborne botulism is a neuroparalytic disease caused by ingestion of foods contaminated with botulinum neurotoxin (BoNT), produced by Clostridium botulinum. In 1995 a husband and wife from Québec, Canada, were hospitalized for several months with prolonged muscle paralysis after ingesting a commercial pâté de campagne. Examination of faecal samples from both patients and the pâté produced viable Group I (proteolytic) C. botulinum type B from each of the three samples. Whole genome sequencing revealed that all three isolates contain identical bont/B5 and bont/F2 genes encoded on a plasmid. Both faecal isolate genomes were identical in chromosome and plasmid length, as well as gene content. The genome of the pâté isolate was nearly identical to that of the faecal isolates with the notable difference of a missing 13-gene insertion on the bont/B5 cluster disrupting the ntnh gene. Examination of the insertion revealed several mobile genetic elements that participate in recombination.

Exploring the Off-label Use of Botulinum Toxin in Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a therapeutic challenge despite various treatment modalities. This review explores the potential of botulinum toxin, both type A (BTX-A) and type B (BTX-B), as an off-label intervention for hidradenitis suppurativa management. Hidradenitis suppurativa, characterized by inflammatory lesions and abscesses, often leads to chronic pain and poor quality of life. Conventional therapies offer limited sustained relief, prompting exploration into alternative treatments. Existing research, although primarily consisting of case reports and a limited randomized controlled trial, suggests potential benefits of botulinum toxin in alleviating HS symptoms. Observations include improvements in dermatology life quality index (DLQI), pain reduction, and lesion regression following botulinum toxin administration. However, these findings lack uniformity in dosing and demographic representation, necessitating further robust investigations encompassing diverse populations. While initial findings hint at the promise of botulinum toxin as a potential therapeutic avenue for HS, the current evidence base is mainly anecdotal. Large-scale controlled trials across diverse demographics are crucial to substantiate efficacy, establish standardized treatment protocols, and evaluate the safety profile for broader application in managing this challenging condition.

Pain-Reducing Modalities Before Botulinum Type A Injections for Hemifacial Spasm Treatment: A Comparative Study

Background: Botulinum toxin A (Botox) therapy is safe and effective for hemifacial spasm, however, the associated discomfort and pain from injections limit its usefulness. Objective: To evaluate pain, preparation difficulty, and satisfaction following application of a eutectic mixture of local anesthetic (EMLA) cream, ice application, and placebo before Botox injections for hemifacial spasm. Materials and Methods: Twenty-two patients received four Botox injections in the facial area to treat hemifacial spasm, between June 2022 and March 2023. The initial treatment began in June 2022, followed by treatments at 3, 6, and 9 months. The EMLA cream, ice application, control, and placebo were administered consecutively for each Botox treatment. Pain, preparation difficulty, and satisfaction were evaluated on a 0 to 10 scale. Neurological and bleeding complications were recorded. Results: The mean pain score for EMLA cream application was the lowest, followed by cold compressions. The mean pain score in the control with placebo intervention was not significantly different from that in the control. The control without placebo intervention had the lowest difficulty in preparation score at 0.82 and showed no significant difference with EMLA application. Whereas cold compression had the highest difficulty in preparation score. The highest satisfaction score was obtained with the EMLA cream application at 9.45. The satisfaction score was higher in the control with placebo intervention at 8.32 than in the control, which had the lowest satisfaction score at 6.91. The number of injections and neurological complications did not differ among the four methods. The bleeding site ratio was the lowest with the cold compression method. Conclusion: EMLA cream application resulted in the lowest mean pain and the highest satisfaction scores. Control with placebo intervention increased the satisfaction score without reducing the mean pain score. Cold compression decreased bleeding complications, and the secondlowest mean pain scores. Keywords: Ice; Local anesthetics; EMLA; Hemifacial spasm; Botox injection; Pain reduction

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures.

Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in Clostridium botulinum type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of C. botulinum. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.

Long-gap esophageal atresia: management, most frequent complications, and expert recommendations – review of literature

Introduction and aim of the study. Long-gap esophageal atresia (LGEA) is a congenital anomaly in which the gap between both ends of the esophagus exceeds three intervertebral spaces and is an esophageal atresia without air in the abdomen. The defect is both therapeutic and surgical challenge. This review aims at providing an overview of the most recent literature on the effective methods for treatment of LGEA, and the most frequent complications and experts’ recommendations on this subject.
 Material and methods. The systematic review was based on available data collected using PubMed database and the Google Scholar web search engine.
 Analysis of the literature. There is no consensus on the ideal technique for surgical treatment of LGEA. There are two possible approaches for opening the thorax – thoracotomy and thoracoscopy. The techniques stimulating esophageal elongation include external and internal traction techniques, magnetic compression anastomosis and intramural botulinum type A toxin injection. Replacement methods are a viable option when it is impossible to preserve the native esophagus. Decellularized matrices seem to be promising in developing an esophageal substitute. Regardless of the surgical approach a common complication of surgical treatment is anastomotic stenosis which requires further surgical interventions.
 Conclusion. Elongation techniques are effective in approximation of the proximal and distal esophagus. The future lies with tissue engineering and inventing an off-the-shelf esophageal substitute. The centralization of treatment is recommended. After discharge from hospital interdisciplinary outpatient assessment and care is required. Further prospective studies are needed to determine the optimal mode of treatment and prevent complications associated with LGEA.

Presence of Clostridium spp. and Clostridium botulinum Types A, B, E, and F in Honey

Presence of Clostridium spp. and Clostridium botulinum Types A, B, E, and F in Honey

Physical Treatments to Control Clostridium botulinum Hazards in Food.

Clostridium botulinum produces Botulinum neurotoxins (BoNTs), causing a rare but potentially deadly type of food poisoning called foodborne botulism. This review aims to provide information on the bacterium, spores, toxins, and botulisms, and describe the use of physical treatments (e.g., heating, pressure, irradiation, and other emerging technologies) to control this biological hazard in food. As the spores of this bacterium can resist various harsh environmental conditions, such as high temperatures, the thermal inactivation of 12-log of C. botulinum type A spores remains the standard for the commercial sterilization of food products. However, recent advancements in non-thermal physical treatments present an alternative to thermal sterilization with some limitations. Low- (<2 kGy) and medium (3-5 kGy)-dose ionizing irradiations are effective for a log reduction of vegetative cells and spores, respectively; however, very high doses (>10 kGy) are required to inactivate BoNTs. High-pressure processing (HPP), even at 1.5 GPa, does not inactivate the spores and requires heat combination to achieve its goal. Other emerging technologies have also shown some promise against vegetative cells and spores; however, their application to C. botulinum is very limited. Various factors related to bacteria (e.g., vegetative stage, growth conditions, injury status, type of bacteria, etc.) food matrix (e.g., compositions, state, pH, temperature, aw, etc.), and the method (e.g., power, energy, frequency, distance from the source to target, etc.) influence the efficacy of these treatments against C. botulinum. Moreover, the mode of action of different physical technologies is different, which provides an opportunity to combine different physical treatment methods in order to achieve additive and/or synergistic effects. This review is intended to guide the decision-makers, researchers, and educators in using physical treatments to control C. botulinum hazards.

Discovery of novel virulence mechanisms in Clostridium botulinum type A3 using genome-wide analysis

ObjectiveClostridium botulinum type A is a neurotoxin-producing, spore-forming anaerobic bacterium that causes botulism in humans. The evolutionary genomic context of this organism is not yet known to understand its molecular virulence mechanisms in the human intestinal tract. Hence, this study aimed to investigate the mechanisms underlying virulence and pathogenesis by comparing the genomic contexts across species, serotypes, and subtypes. MethodsA comparative genomic approach was used to analyze evolutionary genomic relationships, intergenomic distances, syntenic blocks, replication origins, and gene abundance with phylogenomic neighbors. ResultsType A strains have shown genomic proximity to group I strains with distinct accessory genes and vary even within subtypes. Phylogenomic data showed that type C and D strains were distantly related to a group I and group II strains. Synthetic plots indicated that orthologous genes might have evolved from Clostridial ancestry to subtype A3 strains, whereas syntonic out-paralogs might have emerged between subtypes A3 and A1 through α-events. Gene abundance analysis revealed the key roles of genes involved in biofilm formation, cell–cell communication, human diseases, and drug resistance compared to the pathogenic Clostridia. Moreover, we identified 43 unique genes in the type A3 genome, of which 29 were involved in the pathophysiological processes and other genes contributed to amino acid metabolism. The C. botulinum type A3 genome contains 14 new virulence proteins that can provide the ability to confer antibiotic resistance, virulence exertion and adherence to host cells, the host immune system, and mobility of extrachromosomal genetic elements. ConclusionThe results of our study provide insight into the understanding of new virulence mechanisms to discover new therapeutics for the treatment of human diseases caused by type A3 strains.

RECONSTRUCTION AND ANALYSIS OF THE TRANSCRIPTOME REGULATORY NETWORK OF CLOSTRIDIUM BOTULINUM TYPE A3 STR. LOCH MAREE

Clostridium botulinum type A3 str. Loch Maree is a clinically important strain that produces botulinum neurotoxin type A3 and causes foodborne, infant, and wound botulism worldwide. Studying the mechanism underlying the virulence of this organism is imperative to understand its antibacterial resistance and discovering new drugs or inhibitors. The biochemical and molecular characteristics of this organism have been intensively studied, but their gene regulatory mechanisms are unclear. Hence, we reconstructed the transcriptional regulatory network from the complete genome of this organism and analyzed interactive genes from the identified hub module using a knowledge-based bottom-up approach. The biological reliability, topological properties, and robustness of the regulatory network model were validated with network parameters, followed by gene ontology terms and literature support. The reconstructed regulatory network consisted of 12 transcriptional regulators associated with 2369 coding genes. ResD, SpoOA, ComK, CcpC, DinR, DegU, CitT, CodY, GerE, GltC, GltR, IolR, and LevR were identified as transcriptional regulators from this organism homologous to Bacillus subtilis 168. These regulators have been shown to control beta-lactamase, methyl-accepting chemotaxis protein, DNA replication protein DnaD, sensor histidine kinase, and putative membrane proteins of this organism. This study also predicted all possible promoter sites in regulated genes and their associated molecular functions. We conclude that a global regulatory network model of this organism provides insights into its growth physiology and virulence elicitation in the human intestinal environment.

The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits

Background: Botulinum toxin (BoNT) or Botox injections are popular non-surgical and non-invasive option to optimize and change an individual’s facial appearance and achieve rejuvenation. Pyridostigmine is an acetylcholinesterase inhibitor important in clinical practice. This study evaluated the toxic effect of BoNT to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine. Methods: We conducted an experimental interventional study to evaluate the antidote effect of pyridostigmine against BoNT. Forty rabbits (skeletally mature males, body weight 1000–2000 g) were included in the study. The rabbits were injected with Clostridium botulinum type A neurotoxin complex (BoNT or Botox), which was reconstituted with 0.9% sterile sodium chloride to a concentration of 100 U/2.5 mL. Animals were divided randomly into four groups (10 in each) as follows: Group 1 (control group), group 2 (pyridostigmine-treated group), group 3 (BoNT only group), and group 4 (BoNT + pyridostigmine treated group). Results: In the control group, there was statistical difference between the rabbits’ weight (P=0.03). The left muscle was significantly heavier than the right (P=0.025). In the pyridostigmine only group, the rabbits’ weight had a high statistical difference during four weeks of the study (P=0.002). In the BoNT injection group, weight had high statistically significant difference four weeks of the study (P=0.033), which was more in first week (1.55±0.2 kg) and then subsequently decreased in the fourth week (1.345±0.13 kg). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was significantly lower than the mean weight of the rabbits’ left quadriceps muscle (8.09±1.2 g, P=0.0001). Conclusions: To the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight of rabbits in comparison to rabbits that received BoNT injections. Pyridostigmine can act as strong antidote against Botox toxicity.

Effect of Intragastric Botulinum Type A Injection Combined with a Low-Calorie High-Protein Diet in Adults with Overweight or Obesity.

(1) Background/aims: Intragastric botulinum toxin A injection (IGBI) combined with diet control is a new and effective weight loss method for grade 2 obese patients. However, the application of IGIB on overweight or obese adults still needs further research to confirm its efficacy. (2) Methods: We retrospectively collected medical data from 1 July 2021 to 1 January 2022 from a total of 71 patients without diabetes who participated in the bariatric clinic with a body mass index (BMI) > 25 kg/m2. Forty-nine participants opted for intragastric botulinum injection (IGBI) using 300 units of botulinum injected into the antrum, body, and fundus, followed with a low-calorie high-protein diet course. Another 22 people participated only in the low-calorie high-protein diet course as a placebo group. This study analyzes the weight loss percentage of the two groups. Adverse events after IGBI are also reported in a safety assessment. (3) Results: In terms of the characteristics of the two groups, the mean BMI was 29.3 kg/m2 in the IGBI group and 28.0 kg/m in the placebo group (p = 0.63 without significant difference). Comparing the percent weight loss from baseline in the two groups after 12 weeks, the IGBI group lost 11.5% of their body weight and the placebo group lost 1.8%. In terms of group analysis, the percentages of participants with a weight reduction of at least 5% for the IGBI and placebo groups were 95% and 4%, respectively. For weight reduction of at least 10%, these values for the IGBI and placebo groups were 63% and 4%, respectively. In terms of adverse events after IGBI for 12 weeks, 12 participants (24.4%) had constipation, which was the main side effect. No serious adverse events were observed during the study period. (4) Conclusion: The combination of a low-calorie high-protein diet and IGBI is an effective and safe procedure in overweight or obese adults for weight reduction, but further larger studies are needed.

Detection and inhibition of Clostridium botulinum in some Egyptian fish products by probiotics cell-free supernatants as bio-preservation agents

The detection and inhibition of Clostridium botulinum by probiotics' cell-free supernatants, as a bio-preservation strategy, were investigated in some Egyptian fish-stuffs i.e., canned sardines, fesikh, moloha, renga, and anchovy. The conventional and multiplex polymerase chain reaction were used to detect C. botulinum, while the inhibitory and antioxidant capacity of four probiotics' cell-free supernatants were assessed using disc diffusion assay (DDA) and radical scavenging activity approaches, respectively. The results revealed that the C. botulinum was isolated from 41.6% of the examined products, where the type E. was the most predominant genotyping. The DDA showed that Lactobacillus bulgaricus EMCC 1102 and L. paracasei EMCC 1103 expressed the highest inhibitory action against C. botulinum type E. Meanwhile, L. bulgaricus EMCC 1102 has higher in vitro antioxidant activity (IC50) than L. paracasei EMCC 1103, mostly due to its high bioactive constituent. Consequently, L. bulgaricus EMCC 1102 was used to control the growth of C. botulinum type E in stored anchovy fillets. L. bulgaricus EMCC 1102 supernatant showed a dose-dependent inhibitory effect on C. botulinum type E. These data provided an innovative perspective on the inhibitory potential of probiotics, as a bio-preservation strategy, to guarantee fish products safety for further applications in the food industry.

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

Tracing investigation and analysis of a Clostridium botulinum food poisoning incident in Bayingolin Mongolian Autonomous Prefecture, Xinjiang

To analyze a suspected case of Clostridium botulinum food poisoning in Bayingolin Mongolian Autonomous Prefecture, Xinjiang and to help validating the diagnosis and providing technical support for clinical treatment. The basic information and clinical manifestations of food poisoning cases were investigated by using the epidemiological method of food safety accidents. The botulinum toxin genes in the samples were detected by real-time PCR and inoculation of KM mouse. The enriched bacteria were further purified and validated. PFGE and cluster analysis were performed on five isolates. Clostridium botulinum type A was detected in two homemade fermented bean samples and stool lavage fluid samples of three patients from enriched samples by toxin test and real-time PCR, and were further validated after isolation of Clostridium botulinum. PFGE showed 100% homology among five isolates. Five isolates of bacteria isolated from the stool lavage fluid of three patients and two homemade fermented bean curd were identified as the same source through PFGE. The cause of this food poisoning cases is food pollution of Clostridium botulinum type A.

Predictive model for growth of Clostridium botulinum from spores at temperatures applicable to cooling of cooked ground pork

Cooling deviations and temperature abuse are two main reasons leading to the risk of Clostridium botulinum outgrowth in cooked pork. The aim of this research was to create a model that could be used to estimate C. botulinum growth from spores in cooked pork at temperatures similar to those used to chill cooked pork in processing facilities and food establishments. A cocktail of proteolytic C. botulinum types A and B consisting of five strains per type were used to inoculate pork to a final spore concentration of approximately 2 log CFU/g and cooked to 71 °C to heat shock the spores and kill vegetative microbes. The growth of C. botulinum was established at constant storage temperatures from 10 to 46 °C. C. botulinum growth was also studied under dynamic temperature conditions with cooling set to start at 54.4 °C and end at 4.4 °C or 7.2 °C in monophasic or biphasic cooling profiles, respectively. Growth parameters were estimated using the Baranyi model as a primary model and growth rates were fitted using the modified Ratkowsky secondary model with respect to temperature. The R2 values ranged from 0.7653 to 0.9995 indicating that the Baranyi primary model was well suited to the growth data. The modified Ratkowsky secondary model's R2 was 0.9653 and its root mean square error (RMSE) was 0.0687. All 11 prediction error values computed were within the limit of acceptable prediction zone (−1.0 to 0.5) suggesting a good fit of the model. The predictive model can provide information for the safety of cooked pork exposed to longer chilling times or for customized process schedule development as cooling of larger diameter products presents a processing challenge in the meat process operations.

Prevalent toxin types of Clostridium botulinum in South Korean cattle farms

Clostridium botulinum produces neurotoxic substrates that can cause fatal flaccid paralysis called botulism. These neurotoxins are classified into types A–G. Several botulism cases were recorded in 2012–2013 in the Gyeonggi province, South Korea. We assessed the distribution of C. botulinum types B, C, and D in several South Korean farms. A total of 184 samples collected in 2012–2013, including feces (n = 72), hay and silage (n = 50), soil (n = 26), water trough (n = 21), and stomach contents (n = 15), were subjected to multiplex polymerase chain reaction (PCR) to screen for types B, C, and D. Twenty-four samples tested PCR-positive as follows: type B (n = 11), type C/D (n = 4), and type D (n = 18). Eight of the 11 type B samples were detected in hay and silage. Sixteen of the 18 type D samples were detected in fecal and stomach content samples. PCR-positivity was observed in fecal (n = 9, 12.5%), hay and silage (n = 10, 20.0%), water trough (n = 2, 9.5%), and stomach content (n = 12, 80.0%) samples. Fourteen (42.4%) C. botulinum-positive samples were isolated from the PCR-positive samples (type B [n = 8], type C/D [n = 1], and type D [n = 5]). Our findings demonstrate that C. botulinum types B, C/D, and D were prevalent in South Korean cattle farms between 2012 and 2013.