Acta Scientiae Veterinariae, 2015. 43: 1333. RESEARCH ARTICLE ISSN 1679-9216 Pub. 1333 Identification of Clostridium perfringens and Salmonella spp. in Swine Through PCR Lívia Boarini1, Mariana Casteleti Beraldo-Massoli1, Mariana Froner Casagrande1, Marita Vedovelli Cardozo1, Lilian Cristina Makino2, Andressa de Souza3, Marco Monteiro de Lima4, Walter Maldonado Jr.5 & Ruben Pablo Schocken-Iturrino1 ABSTRACT Background: The presence Clostridium spp. and Salmonella spp. is the main concern in swine sector, since they interfere in the microbiological quality of meat and can be responsible for several diseases, presenting occurrences such as intestinal infections and enterotoxaemia. It is necessary to improve production and quality of meat and by-products swine ensur- ing good quality origin of products marketed. Considering these aspects, the objective of the present study was to assess the presence of Clostridium perfringens and Salmonella spp. in pigs destined for consumption, with bacterial counts and identification using PCR technique. Materials, Methods & Results: The collections were made at slaughterhouse and confinement, with bacterial count by the pour-plate technique with Reinforced Clostridial Agar, and the identification by PCR with cpa, cpb, IA, cpe, etx and cpb-2 genes for C. perfringens; and invA and fliC for Salmonella spp. The scores presented average values of 6.04 x 104 CFU/mL for confinement, and 1.8 x 104 CFU/mL in the slaughterhouse. The means counts between the confinement and slaughterhouse groups showed significant difference (P < 0.01). The samples taken at the slaughterhouse were from several farms at random, and allowed the appraisal of hygienic conditions, since animals of confinement remained sanitized and the accumulation of feces on floor was avoided. The PCR technique presented positive results forC. perfringens alpha toxin (cpa) in 25% of the confinement samples, and 46.4% in the slaughterhouse.Salmonella spp. was identified in 9.5% of confinement and 21.4% of the slaughterhouse. Discussion: The presence of C. perfringens and Salmonella spp. is directly related to losses in the entire system of cre- ation of pigs, since infected animals are susceptible to severe diseases, or without clinical signs can exhibit apathy and loss of appetite making weight gain difficult and causing deficiency of the immune system. The difference between the averages counts of the confinement and the slaughterhouse may be due to several factors, but environmental conditions and hygienic are the most influence these differences. When the slaughterhouse samples were collected, the contact with swine of the other farm and the stress of transport may allow the excretion of C. perfringens in greater quantity than usual, while the animals of confinement remained in stalls separate and stress-free the whole period. Many researches have been demonstrating that the identification of pathogenic bacteria by PCR has been more sensitive and faster than conventional microbiology. For this work, this technique gave swift and reliable results, since it consisted of amplification of specific genes to the species of C. perfringens and Salmonella spp. and S. Typhimurium. This fact is of great relevance to contribute preventively with the diagnosis and treatment of animals before they can cause damage to the sector. This comparison can be confirmed when observed the correlation coefficient (- 0,25) between detection of bacterial and weight of animals confined. These results demonstrate that animals with high count of bacteria were those who had greater difficulties in gaining weight, and consequently were slaughtered below the ideal weight. It could be concluded that C. perfringens and Salmonella spp. are often related directly to negative influence on pig production, mainly in initial confinement hindering the weight gain of the animals, and in the industry sector contaminating the meat and its derivatives. Moreover, the molecu- lar biology methods showed to be quite efficient when compared to the conventional ones, thus, PCR can be considered a useful tool to improve pig production. Keywords: anaerobic bacteria, swine production, slaughterhouse. Received: 4 June 2015 Accepted: 8 December 2015 Published: 19 December 2015 1Departamento de Patologia Veterinária, 3Departamento de Medicina Veterinária Preventiva, 4Departamento de Zootecnia e 5Departamento de Ciências Exatas, Faculdade de Ciências Agrárias e Veterinárias/Universidade Estadual Paulista (FCAV/UNESP), Jaboticabal, SP, Brazil. 2Departamento de Engenharia de Pesca, Universidade Estadual Paulista (UNESP), Registro, SP, Brazil. CORRESPONDENCE: L. Boarini [[email protected] - Tel.: +55 (16) 3209-7369]. Departamento de Patologia Veterinária, Prédio de Microbiologia, FCAV/UNESP. Via de Acesso Prof. Paulo Donato Castellane s/n. CEP 14.884-900 Jaboticabal, SP, Brazil. 1 L. Boarini, M.C. Beraldo-Massoli, M.F. Casagrande, et al. 2015. Identification of Clostridium perfringens and Salmonella spp. in Swine Through PCR. Acta Scientiae Veterinariae. 43: 1333. INTRODUCTION Methodology The pathogens Clostridium spp. and Salmo- The seek for Clostridium spp. began with se- nella spp. are directly related to diseases in piglets, rial dilutions subjected to thermal shock [4] to active such as intestinal infections and enterotoxaemia. This germination of spores and elimination of vegetative are patogens found in the intestinal tract of animals cells and possible contaminants. and are identified as responsible for a wide range of After thermal shock, an aliquot of 1 mL of diseases in human and animal. each dilution was transferred to a Petri dish, and The pathogenicity of C. perfringens in the in- Reinforced Clostridial Agar was added by the pour testinal tract of pigs is associated whit the occurrence of plate method and incubated in anaerobic conditions 1 toxin, mainly toxins α (type A) and β (type C) [15,17]. with GasPack system at 37°C for 48 h. Typical Which can cause diarrhea, hemorrhagic, necrotic colonies (3 to 5)were inoculated in BHI broth tubes enteritis and malignant edema and is considered the (Brain Heart Infusion)1 and incubated at 35°C for 24 leading cause of diarrhea in newborns. h. DNA extractions were performed by the technique Infections by Salmonella spp. have worldwide of Marmur [12]. dissemination and spreads quickly among animals The PCR reactions were directed for the de- why contact with the stool, causing great economic tection of genes encoding toxins alpha, beta, Epsilon, losses and public health [13]. As well as, failures in iota (Table 1). The reactions followed 1X buffer [100 management at the industrial process carries those mi- mM Tris-HCl pH 8.8; 500 mM KCl; 0.8% (v/v) Non- croorganisms from infected animals during slaughter, idet P40]; MgCl2 2 mM; dNTP’s 0.2 mM, 1.5 U of extending the scale of contamination to the consumer. Taq DNA polymerase, 5 pmol of each primer, 60 ng It is considered of utmost importance the microbio- of genomic DNA and sterile pure water 20 µL. The logical control of these agents to minimize economic amplifications were in thermal cycler Veriti Thermal losses and ensure food and trade security in products Cycler2 programmed with annealing temperature of derived from pork. 55°C for 1 min. The electrophoretic used agarose gel The growing market expansion, care with pro- 1% (p/v) with SYBR Green3 1X and molecular size duction and marketing of meat are essential for better standard 100 pb DNA Ladder4 , and photo documenta- product quality and food safety, sice C. perfringens tion GEL DOCTM EQ5. food poisoning ranks among the most common food- For Salmonella spp., samples were incubated at borne diseases worldwide [11]. 42°C for 24 h, transfer 1 mL in 10 mL Selenite broth- As infected pigs are still a major source of Novobiocin® and Rappaport Vassiladis for enrichment introduction of bacteria into the human food chain at 37°C and 42°C, for 24 h. After enrichment, samples [8], the present study has as objective to evaluate the were isolated on MacConkey Agar and XLD with presence of C. perfringens and Salmonella spp. in incubation at 37°C for 24 h. Suggestive colonies of pigs. Using bacterial counts and molecular biology Salmonella spp. were place onto Radhanjs Agar6, Agar techniques allow rapid and accurate identification Rambach7 and TSI (Triple Sugar Agar) and incubated results. under the same conditions. The extraction of DNA of Salmonella also followed the technique of Marmur MATERIALS AND METHODS [12], and the identification with PCR reactions using oligonucleotides of invA gene (Table 2). Amplifications Sampling were executed in thermal cycler Veriti Thermal Cycler2 Samples were collected in the pig confine- with annealing temperature of 60°C for 1 min. The ment of the College of Agricultural and Veterinarian samples were subjected to electrophoresis in agarose Sciences - UNESP/FCAV, Jaboticabal Campus and in gel 1% (p/v), with SYBR® Green3 1X and molecular slaughterhouse in the countryside of the State of São size standard 100 pb DNA Ladder4, and photo docu- Paulo in Brazil, being 84 animals fom a slaughterhouse mented on GEL DOCTM EQ5. and 84 from the confinement, without signs of infec- Positive samples to invA, were submitted also tion. Anorectal swabs were collected of each animal, to gene fliC (Table 2) in order to identify a possible and placed onto tubes with water peptone 0.1% sterile. Salmonella Typhimurium. The PCR executed in 2 L. Boarini, M.C. Beraldo-Massoli, M.F. Casagrande, et al. 2015. Identification of Clostridium perfringens and Salmonella spp. in Swine Through PCR. Acta Scientiae Veterinariae. 43: 1333. thermal cycler Veriti Thermal Cycler2 with anneal- Statistical analysis ing temperature 52°C for 1 min. The samples were Data are presented as mean values ± sd of du- subjected to electrophoresis in agarose gel 1% (p/v) plicate measurements. Statistical analysis was done by with SYBR® Green3 1X and molecular size standard Tukey´s test, P < 0.01 was considered significant, and 100 pb DNA Ladder4, photo documented with DOCTM the coefficient of variation stabilized by transformation GEL EQ5. of values for log (x + 5).