Next Article in Journal
Validation of an Ultra-Wideband Tracking System for Recording Individual Levels of Activity in Broilers
Previous Article in Journal
Dietary Supplementation of Dried Grape Pomace Increases the Amount of Linoleic Acid in Beef, Reduces the Lipid Oxidation and Modifies the Volatile Profile
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 9
Issue 8
10.3390/ani9080579
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The First Identification and Antibiogram of Clostridium perfringens Type C Isolated from Soil and The Feces of Dead Foals in South Korea

by
Chul Song Park
,
Ji Yong Hwang
and
Gil Jae Cho
*
Laboratory of Equine Medicine, College of Veterinary Medicine and Institute of Equine Medicine, Kyungpook National University, 80, Daehak-ro, Buk-gu, Daegu 41566, Korea
*
Author to whom correspondence should be addressed.
Animals 2019, 9(8), 579; https://doi.org/10.3390/ani9080579
Submission received: 7 May 2019 / Revised: 29 July 2019 / Accepted: 13 August 2019 / Published: 20 August 2019
(This article belongs to the Section Equids)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Clostridium (C.) perfringens in horses causes acute enteritis and death, but research on C. perfringens in South Korea is virtually nonexistent. The purpose of this study was to discover the cause of death of numerous South Korean foals. C. perfringens was isolated from 25 (11.1%) of 225 sampled horses and from 16 (35.56%) of 45 farms investigated in this study. Of the 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all the farms where the foals’ deaths occurred. The antimicrobial susceptibility test was performed using MIC (Minimum Inhibitory Concentration) Evaluator strips test. These results are the first to identify one of the causes of acute foal death in South Korea and can be used as a criterion to determine the cause of acute foal death and to select effective antibiotics.

Abstract

Clostridium (C.) perfringens was isolated from 25 (11.1%) of 225 sampled horses and from 16 (35.56%) of 45 farms. All of the samples were negative for cpe, etx, itx, NetF genes and cpa gene were detected in 100% (25 of 25) of the samples that were positive for C. perfringens. cpb and cpb2 were detected in 40.0% (10 of 25) and 60.0% (15 of 25) of the samples that were positive for C. perfringens, respectively. Of the 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all the farms where the foals’ deaths occurred. None of the isolates were positive for type B, type D, or type E. The MIC Evaluator strips antimicrobial susceptibility test showed meropenem (96%), ampicillin (92%), amoxicillin/clavulanic acid (84%), and tetracycline (8%) sensitivity.

1. Introduction

Clostridium (C.) perfringens is an anaerobic, endospore-forming, gram-positive, rod-shaped bacterium that is ubiquitous in various environments, especially in soil and the gastrointestinal tract of humans and animals [1,2,3,4,5,6]. C. perfringens causes numerous gastrointestinal infections in most mammalian species. These infections are generically called enterotoxemia because toxins produced in the intestine may be absorbed into general circulation [7,8]. C. perfringens produces at least 17 different toxins and, according to the production of the major toxins alpha, beta, epsilon, and iota, it has been classified into five different [8,9] types. Although the diseases caused by each toxin are somewhat different, we hypothesized that the beta-toxin of C. perfringens type C can cause death in foals. C. perfringens type C produces the highly necrotizing and lethal beta-toxin, which is responsible for severe intestinal necrosis. The beta-toxin is encoded by the cpb gene, which is located on a plasmid [3]. The beta-toxin induces lethal infections ranging from necrohemorrhagic enterocolitis to enterotoxemia in pigs, cattle, sheep, and goats and in the neonatal animals of numerous domestic animal species [4,10].
Studies on C. perfringens are currently underway in the world [3,4,5,11,12,13]. In particular, research on C. perfringens type A and its bacterial effects on chickens is abundant, but there are not many studies on C. perfringens type C [1,8,9,14], and those in horses are even rarer [2,15].
C. perfringens type C is an important cause of enteritis and enterocolitis in foals and, occasionally, in adult horses [2]. Because beta-toxin is highly susceptible to the action of trypsin, neonatal animals are particularly susceptible to type C infections due to the low level of intestinal trypsin in the first days of life and the presence of trypsin inhibitors in colostrum [2]. The aim of this study was to identify C. perfringens and its toxin types under the hypothesis that C. perfringens was the cause of severe colitis in numerous foals in South Korea. We also performed antibiotic susceptibility tests for effective disease treatment.

2. Materials and Methods

2.1. Sampling

From 2009 to 2017, 225 horse fecal and soil samples were collected from 45 horse farms located on Jeju island and an inland region of South Korea. Seven samples were collected from the dead foals’ feces (foals who died of acute diarrhea within 3 to 5 days of birth) and contaminated soil around the dead body on each farm. If there were no dead foals on the farm or horse riding track without foals, then samples were collected from healthy horse feces and general soil for comparison (Table 1).
The symptoms of all the dead foals were the acute onset of diarrhea with multiple clinical signs of toxemia, such as fever, depression, dehydration, tachycardia, prolonged capillary refill time, lying down, congested mucous membranes, and multifocal hemorrhagic inflammations of the intestine identified by postmortem examination. These symptoms appeared within 5 days of the foal’s birth (data not presented here).
The collected samples were stored in sterile plastic tubes and frozen at −70 °C within 6 to 12 h for later analysis [16,17,18,19].

2.2. C. perfringens Detection

An attempt was made to isolate the causative bacteria from the samples of suspected farms to diagnose whether the cause of the foals’ deaths was C. perfringens. Samples were identified according to the previous study method [20]. First, 10 g of fecal or soil samples were collected in a new tube and diluted with 10 mL of 0.1 M phosphate-buffered saline (PBS, pH 7.4) (Gibco, Dublin, Ireland). The diluted tubes were shaken for 30 min using a shaking incubator (Vision Scientific, Seoul, South Korea). The supernatant (1.5 mL) was transferred to an Eppendorf tube and centrifuged at 1000 rpm for 5 min (Labogene, Seoul, South Korea). After centrifugation, 1 mL of the supernatant was taken out, transferred to another Eppendorf tube, and centrifuged at 8000 rpm for 10 min. After that, 1 mL of the supernatant was removed and 1 mL of PBS was added, transferred to a dry bath (Biometra, Dublin, Ireland), and heat treated at 80 °C for 10 min. The heat-treated sample (1 mL) was inoculated into cooked meat medium (CMM) (Oxoid, Hampshire, UK) and incubated in an anaerobic jar ( Rectangular jar, AnaeroPack ™, Thermoscientific, Massachusetts, US) at 37 °C for 2 days in an incubator (Panasonic, Osaka, Japan). The cultured CMM medium was shaken well, sub-cultured onto blood agar plates, and anaerobically incubated for 2 days at 37 °C. Two days later, the C. perfringens colonies were evaluated for the large double zone of hemolysis and microscopic morphology characteristics. These isolates were characterized using VITEK® MS (Biomerieux, Marcy-l’etoile, France) according to the manufacturer’s instructions. They were stored in Microbank (Prolab, Texas, US) and further characterized as described in a later section.

2.3. Preparation of Genomic DNA

The DNA extraction of C. perfringens requires the extraction of its plasmid genes along with the chromosomal genes, because most of the toxin genes are derived from plasmids. Therefore, three DNA extraction methods were performed to obtain accurate results. The first was the boiling method in which one or two single colonies were added to 100 μL of distilled water or PBS, shaken thoroughly with sterilized platinum, heat-treated at 100 °C for 30 min in a dry bath, and centrifuged at 13,000 rpm for 5 min. PCR was performed using 50 μL of the supernatant [13,21]. Two other methods were performed using commercial DNA kits: Qiamp DNA mini kit (Qiagen, Hilden, Germany) and Qiaprep spin miniprep kit (Qiagen, Hilden, Germany), according to the manufacturers’ instructions.

2.4. 16S rRNA Gene Sequencing and Statistical Analysis

16S rRNA gene sequencing was performed for a more accurate identification of all the strains primarily identified as C. perfringens [22,23] with an ABI PRISM 3730XL Analyzer (Applied Biosystem, Foster City, CA, USA). The alignment was performed with BioEdit, and the nucleotide sequences of the strains were aligned using the ClustalW multiple alignment function with a bootstrap value set to 1000. The phylogenetic tree of the isolated strains was analyzed using 11 standard strains from NCBI (National Center for Biotechnology Information) using the neighbor joining tree method by MEGA 6.0 software program [24,25].

2.5. C. perfringens Toxin Detection

All strains identified primarily as C. perfringens were examined for alpha toxin (cpa) genes and enterotoxin (cpe) genes using commercially available real-time PCR kits (Kogenbiotech, Seoul, Korea), according to the manufacturers’ instructions. The remaining beta toxin (cpb), epsilon toxin (etx), iota toxin (itx), beta2 toxin (cpb2) and NetF genes were identified by the conventional PCR method. Detection of toxin genes was carried out using primers, probes, and conditions described in a previous study [26].

2.6. Antibiogram Test

The antimicrobial susceptibility testing was performed using the MIC Evaluator strips test (E-test) with eight antibiotics (penicillin, amoxicillin/clavulanic acid, ampicillin, tetracycline, vancomycin, meropenem, clindamycin, and metronidazole) according to previous study methods [27,28,29].

3. Results

3.1. C. perfringens Detection

Twenty-five (11.1%) C. perfringens was isolated from 225 samples tested and 16 (35.56%) of 45 farms. These 25 strains were 100% identical to the nucleotide sequence of C. perfringens (ATCC 13124) registered on the NCBI website by using VITEK analysis and 16S rRNA gene sequencing (Figure 1, Table 1).

3.2. Toxins and Types of C. perfringens

All the samples were negative for cpe, etx, itx, NetF genes and cpa gene was detected in 100% (25 of 25) of the samples that were positive for C. perfringens. cpb and cpb2 were detected in 40.0% (10 of 25) and 60.0% (15 of 25) of the samples that were positive for C. perfringens, respectively (Figure 2, Table 2). Of the 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all seven farms where the foals’ deaths occurred and horse riding tracks at three farms without foals present. None of the isolates were positive for type B, type D, or type E.

3.3. Antibiogram Test

The results of the antimicrobial susceptibility testing of C. perfringens is shown in Table 3. Briefly, the MIC E-test showed meropenem (96%), ampicillin (92%), amoxicillin/clavulanic acid (84%), and tetracycline (8%) sensitivity.

4. Discussion

Due to the extensive size of their colon, horses are more seriously affected by colitis than other animals without an expanded hindgut. Acute enterocolitis in horses is known mainly as Clostridium difficile, C. perfringens, larval cyathostomiasis, Neorickttsia risticii, and Salmonella [30]. Among these infectious agents, C. difficile and Salmonella are known to be major contributors to acute colitis in horses. Although progress has been made in identifying the causes of acute colitis in horses, the cause is still unknown in approximately 60% of cases [19]. We hypothesized that C. perfringens and its production of enterotoxin (cpe) and/or beta2 toxin (cpb2) are common and important causes of severe colitis in horses [19].
Of 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all the farms where the foals’ deaths occurred. However, it is difficult to conclude that type C is the direct cause of death of the foals. Also, the plasmid DNA extraction has not been carried out perfectly. There are correlation between this experiment and the mortality of the foals. Studies on C. perfringens have been carried out in humans and animals, and those of type A, cpe, or cpb2 genes are common. In the case of humans, studies about Clostridium botulinum and food poisoning by the cpe (enterotoxin) of C. perfringens type A, which is a common cause of food poisoning, are progressing. In animals, studies are mainly performed on type A, cpb2, PfoA, and NetB toxins in pigs and chickens [21,22,31,32]. Enterotoxin (cpe) was not detected in any of the isolates. The detection of cpe gene varies among researchers as well as geographical differences and seasonal variations. This is the first time that the cpe gene has been investigated in C. perfringens isolated from equine enterocolitis in South Korea. Type C infections have occasionally been recorded as a cause of colitis in adult horses [2]. There was no difference between foals and adults in clinical presentation.
The inclusion of foals is another possible limitation of this study since foals may suffer from viral infections or C. perfringens necrotic enteritis toxin B (NetB), which could cause a foal’s death, although none were recorded. Autopsies after the deaths of the foals were only conducted on the macro and pathological failures, which also limits the definitive determination of C. perfringens as the cause of death of the foals. However, based on this sampled population and acknowledging the limitations of this study, including the absence of a healthy comparison population, we speculate that the cause of death of the foals has considerable relevance to C. perfringens type C in South Korea as reported in North America [2,15]. Extensive efforts, including occurrence epidemiology, will be required to identify the unknown causes of severe colitis in horses due to the complexities of case selection, isolating and identifying potential pathogens, and assessing toxin production. In particular, extensive investigations of cpb2 and NetB genes, which are the major causes of enteritis, irrespective of the type of C. perfringens, are further required.
Studies are underway to find susceptible antibiotics to treat animals infected with C. perfringens [33]. However, antibiotic susceptibility studies on C. perfringens in horses in South Korea are limited. Therefore, we performed antibiotic susceptibility tests using 25 C. perfringens strains in this study and found that meropenem and ampicillin significantly affected the C. perfringens strains. Because C. perfringens is a gram-positive bacterium, antibiotics of the carbapenem and penicillin family, which inhibit cell wall synthesis, act strongly. The E-test method has the advantage of obtaining more detailed information because a concentration of 0.002 µg/mL can be used, which is lower than the minimum dilution factor for the general broth dilution method (0.016 µg/mL). This antibiotic test showed no difference in the antibiotic susceptibility of C. perfringens according to its type and toxin. Antimicrobial susceptibility testing is very important in the treatment of bacterial diseases. Clearly, vaccination is the most useful method to prevent disease, but if the vaccine is not available, adequate antibiotic treatment in the early stages of the disease may be the next maximized option. Especially in cases of gastrointestinal diseases that affect productivity, rapid treatment must be performed to reduce economic loss. Also, since C. perfringens type C infections may cause mortality within one to two days after birth, antibiotics should be considered as a preemptive strategy on farms where foal acute death occurs frequently.

5. Conclusions

The purpose of this study was to discover the cause of death of numerous foals in South Korea. C. perfringens was isolated from 25 (11.1%) of the 225 horses sampled in this study and from 16 farms (16/45, 35.56%). Of the 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all the farms where the foals’ deaths occurred. These results are the first to identify one of the causes of acute foal death in South Korea and can be used as a criterion to determine the cause of acute foal death and to select effective antibiotics. Extensive efforts, including the occurrence epidemiology, will be required to identify the unknown causes of severe colitis in horses due to the complexities of case selection, isolating and identifying potential pathogens, and assessing toxin production.

Author Contributions

Conceptualization, formal analysis, investigation, methodology, and writing the original draft, C.S.P. and J.Y.H.; supervision and writing, reviewing, and editing, G.J.C.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Deguchi, A.; Miyamoto, K.; Kuwahara, T.; Miki, Y.; Kaneko, I.; Li, J.; McClane, B.A.; Akimoto, S. Genetic Characterization of Type A Enterotoxigenic Clostridium perfringens strains. PLoS ONE 2009, 4, e5598. [Google Scholar] [CrossRef] [PubMed]
  2. Diab, S.S.; Kind, H.; Moore, J.; Shahriar, M.F.; Odani, J.; Anthenill, L.; Songer, J.G.; Uzal, F.A. Pathology of Clostridium perfringens Type C Enterotoxemia in Horses. Vet. Pathol. 2012, 49, 255–263. [Google Scholar] [CrossRef] [PubMed]
  3. Li, J.; Adams, V.; Bannam, T.L.; Miyamoto, K.; Garcia, J.P.; Uzal, F.A.; Rood, J.I. Toxin Plasmids of Clostridium perfringens. Microbiol. Mol. Biol. Rev. 2013, 77, 208–233. [Google Scholar] [CrossRef] [PubMed]
  4. Nagahama, M.; Ochi, S.; Oda, M.; Miyamoto, K.; Takehara, M.; Kobayashi, K. Recent Insights into Clostridium perfringens Beta-Toxin. Toxins 2015, 7, 396–406. [Google Scholar] [CrossRef] [PubMed]
  5. Othani, K.; Shimizu, T. Regulation of Toxin Production in Clostridium perfringens. Toxins 2016, 8, 207. [Google Scholar]
  6. Uzal, F.A.; Vidal, J.E.; McClane, B.A.; Gurjar, A.A. Clostridium perfringens toxins involved in mammalian veterinary diseases. Open Toxinol. J. 2014, 2, 24. [Google Scholar] [CrossRef]
  7. Bokori-Brown, M.; Savva, C.G.; Fernandes da costa, S.P.; Naylor, C.E.; Basak, A.K.; Titball, R.W. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS. J. 2011, 278, 4589–4601. [Google Scholar] [CrossRef]
  8. Nakano, V.; Ignacio, A.; Lianco, L.; Bueris, V.; Sircili, M.P.; Avila-Campos, M.J. Multiocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeLand netB genes. Anaerobe 2017, 44, 99–105. [Google Scholar] [CrossRef]
  9. Coursodon, C.F.; Glock, R.D.; Moore, K.L.; Cooper, K.K.; Songer, J.G. TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks. Anaerobe 2012, 18, 117–121. [Google Scholar] [CrossRef]
  10. Songer, J.G. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 1996, 9, 216. [Google Scholar] [CrossRef]
  11. Gkiourtzidis, K.; Frey, J.; Bourtzi-Hatzopoulou, E.; IIiadis, N.; Sarris, K. PCR detectionand prevalence of alpha-, beta-, beta2-, epsilon-, iota-, and enterotoxin genes in Clostridium perfringens isolated from lambs with Clostridial dysentery. Vet. Microbiol. 2001, 82, 39. [Google Scholar] [CrossRef]
  12. Klassen, H.L.; Molkenboer, M.J.; Bakker, J.; Miserez, R.; Hani, H.; Frey, J.; Popoff, M.R.; Van den bosch, J.F. Detection of the beta2 toxin gene of Clostridium perfringens indiarrhoeic piglets in The Netherlands and Switzerland. FEMS. Immunol. Med. Microbiol. 1999, 24, 325–332. [Google Scholar]
  13. Mahony, D.E.; Clark, G.A.; Stringer, M.F.; MacDonald, M.C.; Duchesne, D.R.; Mader, J.A. Rapid extraction of plasmids from Clostridium perfringens. Appl. Environ. Microbiol. 1986, 51, 521–523. [Google Scholar] [PubMed]
  14. Chalmers, G.; Bruce, H.L.; Hunter, D.B.; Parreira, V.R.; Kulkarni, R.R.; Jiang, Y.F.; Prescott, J.F.; Boerlin, P. Multlocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations. J. Clin. Microbiol. 2008, 46, 3957–3964. [Google Scholar] [CrossRef] [PubMed]
  15. Howard-Martin, M.; Morton, R.; Qualls, J.C.; MacAllister, C.G. Clostridium perfringens type C enterotoxemia in a newborn foal. J. Am. Vet. Med. Assoc. 1986, 189, 564–565. [Google Scholar]
  16. Bukar, A.; Mukhtar, M.D.; Adam, S.A. Current trend in antimicrobial susceptibility pattern of Clostridium tetani isolated soil samples Kano. Bayero J. Pure Appl. Sci. 2008, 1, 112–115. [Google Scholar] [CrossRef]
  17. Sanada, I.; Nishida, S. Isolation of Clostridium tetani from soil. J. Bacteriol. 1965, 89, 626–629. [Google Scholar]
  18. Smith, L. Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil. Appl. Microbiol. 1975, 30, 319–323. [Google Scholar] [PubMed]
  19. Gohari, I.M.; Arroyo, L.; MacInnes, J.I.; Timoney, J.F. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis. Can. J. Vet. Res. 2014, 78, 1–7. [Google Scholar]
  20. Murdoch, D.A. Gram-Positive Anaerobic Cocci. Clin. Microbiol. Rev. 1998, 11, 81–120. [Google Scholar] [CrossRef] [Green Version]
  21. Yoo, S.Y.; Lee, S.U.; Park, K.Y.; Park, Y.H. Molecular Typing and Epidemiological Survey of Prevalence of Clostridium perfringens Types by Multiplex PCR. J. Clin. Microbiol. 1997, 35, 228–232. [Google Scholar]
  22. Kalia, V.C.; Mukherjee, T.; Bhushan, A.; Joshi, J.; Shankar, P.; Huma, N. Analysis of the unexplored features of rrs(16S rDNA) of the Genus Clostridium. BMC Genom. 2011, 12, 18. [Google Scholar] [CrossRef] [PubMed]
  23. Woo, P.C.; Lau, S.K.; Chan, K.M.; Fung, A.M.; Tang, B.S.; Yuen, K.Y. Clostridium bacteraemia characterized by 16S ribosomal RNA gene sequencing. J. Clin. Pathol. 2005, 58, 301–307. [Google Scholar] [CrossRef] [PubMed]
  24. Fallani, M.; Rigottier-Gois, L.; Aguliera, M.; Bridonneau, C.; Collignon, A.; Edwards, C.A.; Corthier, G.; Dore, J. Clostridium difficle and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J. Microbiol. Methods. 2006, 67, 150–161. [Google Scholar] [CrossRef] [PubMed]
  25. Hwang, J.Y.; Cho, G.J. First Identification of Taylorella equgenitalis From Genital Tracts of Thoroughbred Horses from the Inland Area of South Korea by Multilocus Sequence Typing. J. Equine Vet. Sci. 2018, 60, 16–22. [Google Scholar] [CrossRef]
  26. Farzan, A.; Kircanski, J.; Delay, J. An investigation into the association between cpb2-encoding Clostridum perfringens type A and diarrhea in neonatal piglets. Can. J. Vet. Res. 2013, 77, 45–53. [Google Scholar] [PubMed]
  27. Andrews, J.M.; Howe, R.A. BSAC standardized disk susceptibility testing method (version 10). J. Antimicrob. Chemother. 2011, 66, 2726–2757. [Google Scholar] [CrossRef] [PubMed]
  28. Nachnani, S.; Scuteri, A.; Newman, M.G.; Avanessian, A.B.; Lomeli, S.L. E-test: A new technique for antimicrobial susceptibility testing for periodontal microorganisms. J. Periodontol. 1992, 63, 576–583. [Google Scholar] [CrossRef]
  29. Thornsberry, C. NCCLS Standards for Antimicrobial Susceptibility Tests. Lab. Med. 2016, 14, 549–553. [Google Scholar] [CrossRef]
  30. Feary, D.J.; Hassel, D.M. Enteritis and colitis in horses. Vet. Clin. North. Am. Equine Pract. 2006, 22, 437–479. [Google Scholar] [CrossRef]
  31. Lee, K.E.; Lim, S.I.; Shin, S.H.; Kwon, Y.K.; Kim, H.Y.; Song, J.Y.; An, D.J. Distribution of Clostridium perfringens isolates from piglets in South Korea. J. Vet. Med. Sci. 2014, 76, 745–749. [Google Scholar] [CrossRef] [PubMed]
  32. Lee, Y.J. Antimicrobial resistance and molecular characterization of Clostridium perfringens isolated from chicken. J. Prev. Vet. Med. 2016, 40, 71–79. [Google Scholar] [CrossRef]
  33. Slavic, D.; Boerlin, P.; Fabric, M.; Klotins, K.; Zothout, J.K.; Weir, P.E.; Bateman, D. Antimicrobial susceptibility of Clostridium perfringens isolates of bovine, chicken, porcine and turkey origin from Ontario. Can. J. Vet. Res. 2011, 75, 89. [Google Scholar] [PubMed]
Animals 09 00579 g001 550
Figure 1. Phylogenetic tree of C. perfringens isolated from horse feces and contaminated soil. The nucleotide sequences of all 25 new strains (named KSJ strains) are identical to those of the NR_121697.1 C. perfringens (ATCC 13124 C. perfringens standard strain). This tree was constructed by the Neighbor-Joining method, MEGA 6.0 program.
Figure 1. Phylogenetic tree of C. perfringens isolated from horse feces and contaminated soil. The nucleotide sequences of all 25 new strains (named KSJ strains) are identical to those of the NR_121697.1 C. perfringens (ATCC 13124 C. perfringens standard strain). This tree was constructed by the Neighbor-Joining method, MEGA 6.0 program.
Animals 09 00579 g001
Animals 09 00579 g002 550
Figure 2. PCR amplification of the beta-toxin (cpb) from 10 C. perfringens isolates. Lane M: molecular size marker (100bp DNA ladder, Bioneer, Korea); Lane 1: standard strain (ATCC 3626) cpb gene; Lane 2, 13 to 16; negative control; Lane 3 to 12: cpb genes detected from 10 C. perfringens isolates.
Figure 2. PCR amplification of the beta-toxin (cpb) from 10 C. perfringens isolates. Lane M: molecular size marker (100bp DNA ladder, Bioneer, Korea); Lane 1: standard strain (ATCC 3626) cpb gene; Lane 2, 13 to 16; negative control; Lane 3 to 12: cpb genes detected from 10 C. perfringens isolates.
Animals 09 00579 g002
Table
Table 1. Clostridium perfringens identified farms and isolates.
Table 1. Clostridium perfringens identified farms and isolates.
TractNo. of FarmsNo. (%) of C. perfringens Identified FarmsNo. of C. perfringens IsolatesNo. of C. perfringens Type (A/C)
Jeju-Island *174 (23.53)94/5
Gyeongsang-Province *54 (80.00)65/1
Chungcheong-Province21 (50.00)10/1
Gyunggi-Province104 (40.00)32/1
Jeolla-province102 (20.00)32/1
Gangwon-Province *11 (100.0)32/1
Total4516 (35.56)2515/10
* The three tracts where the foal’s death emerged were Jeju Island, Gyeongsang Province, and Gangwon Province.
Table
Table 2. Distribution of cpa, cpb, cpe/etx/itx/NetF, cpb2 genes and types among 25 Clostridium perfringens isolated from horses and tracks.
Table 2. Distribution of cpa, cpb, cpe/etx/itx/NetF, cpb2 genes and types among 25 Clostridium perfringens isolated from horses and tracks.
TypesNo. (%) of C. perfringens IsolatesNo. of Isolates from Dead Foals and Trackscpacpbcpe/etx/itx/NetFcpb2
A15 (60.0)0/015/150/150/1510/15
C10 (40.0)7/310/1010/100/105/10
Total25 (100)7/325/2510/250/2515/25
Table
Table 3. Antimicrobial susceptibility testing of 25 C. perfringens isolated from horses.
Table 3. Antimicrobial susceptibility testing of 25 C. perfringens isolated from horses.
AntibioticsNo. (%) of Isolates
ResistanceIntermediateSusceptible
Penicillin0(0)25(100)0(0)
Amoxicillin/clavulanic acid3(12)1(4)21(84)
Ampicillin0(0)2(8)23(92)
Vancomycin1(4)24(96)0(0)
Tetracycline0(0)23(92)2(8)
Clindamycin0(0)25(100)0(0)
Metronidazole0(0)25(100)0(0)
Meropenem1(4)0(0)24(96)

Share and Cite

MDPI and ACS Style

Park, C.S.; Hwang, J.Y.; Cho, G.J. The First Identification and Antibiogram of Clostridium perfringens Type C Isolated from Soil and The Feces of Dead Foals in South Korea. Animals 2019, 9, 579. https://doi.org/10.3390/ani9080579

AMA Style

Park CS, Hwang JY, Cho GJ. The First Identification and Antibiogram of Clostridium perfringens Type C Isolated from Soil and The Feces of Dead Foals in South Korea. Animals. 2019; 9(8):579. https://doi.org/10.3390/ani9080579

Chicago/Turabian Style

Park, Chul Song, Ji Yong Hwang, and Gil Jae Cho. 2019. "The First Identification and Antibiogram of Clostridium perfringens Type C Isolated from Soil and The Feces of Dead Foals in South Korea" Animals 9, no. 8: 579. https://doi.org/10.3390/ani9080579

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop