2.1. Animals and farm
A total of 3,732 Duroc x (Landrace x Large White) were used. These animals came from Colidex-C® (Vetia Animal Health, Spain) sows that were vaccinated in the two previous pregnancies (2 mL, intramuscular injection at 80th day of gestation; injection in the neck, just behind the ear, using disposable needle 18G x 1-1/2"). Piglets were divided into two experimental groups: Vaccinated Group, with 1,893 animals, vaccinated and revaccinated at days 10 and 20 of life with Colidex-C® (0.5 and 1mL, intramuscular injection in the neck, behind the ear; disposable needle 24G x 3/4"); and Control Group, with 1,869 animals, injected with sterile saline solution at days 10 and 20 of life (0.5 and 1mL, intramuscular injection in the neck, behind the ear; disposable needle 24G x 3/4"). The animals were randomly assigned to each group.
Piglets were castrated in the first week of life (according to the Directive 2008/120/EC) and kept during the trial period without routine medication against enteric pathogens. The study was conducted in a three-sites-production system farm, with breeders, nursery, and finishers in the same geographical area (Province of Toledo, Spain). In the nursery phase, the animals were raised in three barns of 1,000 animals each, subdivided into three modules each. The size of the stalls was 2.7 square meters, which housed 12 piglets each. In the growing and finishing phases, there were 11 barns of 600 animals each, with a free surface area of 8.4 square meters where 12 animals were housed. Animals from the Vaccinated Group and Control Group were housed in the same barn but in different stalls. The stalls had identical size and environmental conditions (stall size, environmental temperature, number of drinkers, and feeders). The animals received feed (meal during nursery and pelleted during finishing), and water ad libitum. The facilities were managed under an all-in all-out system. Once they reached market weight (an average 107.82 Kg body weight), pigs were sent to the slaughterhouse.
2.2. Vaccine
The vaccine used was Colidex-C® (Vetia Animal Health, Spain), which is composed of a suspension of seven strains of E. coli, inactivated with formalin and heat, ensuring the presence of fimbriae antigens F4, F5, F41, F18, and F6; and of toxoids of heat-labile toxin (LT), heat-stable toxin (STa), verotoxin (VT) and hemolysin (Hly), combined with a toxoid of Clostridium perfringens Type C (Table 1). Each 2 ml dose contained 2 x 109 micro-organisms from each of the bacterial strains inactivated with heat, 1.5 x 109 and micro-organisms of each strain inactivated with formol and 300 UI of β (beta) toxoid of C. perfringens. Vaccine lot number used in this study: 192761.
By the time the study was conducted, Vetia Animal Health was named Farco Veterinaria S.A. When the company changed its name, some changes were also made to the composition of the vaccine Colidex-C®. Because of that, the company's current website shows a different composition from the one shown in the present study.
Table 1 - Composition of the vaccine (2ml)
Components of the vaccine
|
Concentration
|
Escherichia coli inactivated (fimbriae F4ac), strain p6
|
≥ 1 RP
|
Escherichia coli inactivated (fimbriae F5), strain p1
|
≥ 1 RP
|
Escherichia coli inactivated (adhesins F5 + F41), strain p10
|
≥ 1 RP
|
Escherichia coli inactivated (adhesin F6), strain p2 and p4
|
≥ 1 RP
|
Escherichia coli inactivated (adhesin F18ab), strain p5
|
≥ 1 RP
|
Escherichia coli inactivated (adhesin F18ac), strain p9
|
≥ 1 RP
|
Escherichia coli, toxoid of heat-labile toxin
|
≥ 10 UI
|
Escherichia coli, toxoid of heat-stable toxin
|
≥ 10 UI
|
Escherichia coli, toxoid of verotoxin
|
≥ 10 UI
|
Escherichia coli, toxoid of hemolysin
|
≥ 10 UI
|
Clostridium perfringens type C, toxoid beta
|
≥ 10 UI
|
Mineral Oil (Marcol 52)
|
0.76 ml
|
Montane 80
|
0.042 ml
|
Montanide 103
|
0.042 ml
|
RP: Relative Potency of the antigen, according to a reference vaccine (Ph. Eur. m 0962)
UI: International Unit
2.3. Sampling
2.3.1. Feces of animals with diarrhea on the farm
Fecal samples were collected from animals with diarrhea, individually or during a collective outbreak. If there was an outbreak at least 12 samples were taken. Samples were taken by swabs with Cary Blair agar gel transport medium (COPAN Diagnostics, Inc. Italy) and kept cold during transportation to the Reference Laboratory specialized in Escherichia coli (LREC) of the Faculty of Veterinary Medicine, University of Santiago de Compostela, Spain.
2.3.2. Intestinal mucosa swabs at slaughter
At slaughter, intestinal jejunum mucosa from five animals from each group was randomly sampled using a swab with Cary Blair agar gel transport medium (COPAN Diagnostics, Inc. Italy) and kept cold during transportation to LREC.
2.3.3. Ileum and colon tissue at slaughter
Fresh samples of ileum and colon were randomly taken at slaughter from 20 animals from each group. They were refrigerated and sent to the Cytogenetics and Genomics Laboratory of the Faculty of Veterinary of the University of Murcia, Spain.
2.4. Samples analysis
2.4.1. Isolation and virulence factor genes of E. coli
The tests performed in the LREC with diarrhea samples and intestinal mucosa swabs are described as follows. Bacterial colonies were grown on lactose-MacConkey agar plates at 37oC for 24 h. An aliquot of confluent growth was inoculated into 30 ml of Luria-Bertani broth, incubated with shaking at 37℃ for 4 h and processed for DNA extraction as described previously [8,9]. Virulence factor genes were detected by conventional PCR (Blanco et al., 2004). Researchers investigated the presence of intimin (eae gene), fimbriae (F4, F5, F6, F18, and F41 genes), hemolysin (Hly), heat-labile toxin (LT), heat-stable enterotoxins (Sta and STb), and verotoxin (VT1 and VT2). Primer sequences are described in Table 2. Amplification products were analyzed by gel electrophoresis (1.5% agarose) and UV transillumination (300 nm).
Table 2 – Characteristics of oligonucleotide sequences used for conventional PCR assays to detect E. coli virulence factor genes.
PCR and samples
|
Virulence factor
|
Oligonucleotide sequence (5'-3')
|
Size of amplified product (bp)
|
Reference
|
Conventional PCR, diarrhea and swabs
|
eae
|
F: GGA ACG GCA GAG GTT AAT CTG CAG
|
775
|
[10]
|
R: GGC GCT CAT CAT AGT CTT TC
|
Hly
|
F: GGTGCAGCAGAAAAAGTTGTAG
|
1551
|
[11]
|
R: TCTCGCCTGATAGTGTTTGGTA
|
LT
|
F: GGCGACAGATTATACCGTGC
|
708
|
[12]
|
R: CCGAATTCTGTTATATATGTC
|
STa
|
F: ATTTTTATTTCTGTATTGTCTTT
|
176
|
[13]
|
R: GGATTACAACACAGTTCACAGCAGT
|
STb
|
F: ATCGCATTTCTTCTTGCATC
|
175
|
[12]
|
R: GGGCGCCAAAGCATGCTCC
|
VT1
|
F: CGCTGAATGTCATTCGCTCTGC
|
302
|
[14]
|
R: CGTGGTATAGCTACTGTCACC
|
VT2
|
F: CTTCGGTATCCTATTCCCGG
|
516
|
[14]
|
R: CTGCTGTGACAGTGACAAAACGC
|
F4
|
F: GGTGATTTCAATGGTTCGGTC
|
773
|
[15]
|
R: ATTGCTACGTTCAGCGGAGCGC
|
F5
|
F: CCAGCGCCCGGCAGTAATGACTGC
|
278
|
[16]
|
R: CCACCATTAGACGGAGCGCGG
|
F6
|
F: GCGCCCGCTGAAAACAACACCAGC
|
467
|
[16]
|
R: GTACCGGCCGTAACTCCACCG
|
F18
|
F: GGTACTGTTGCACCAAGCGG
|
225
|
[16]
|
R: CGACGCCTTAACCTCCTGCCCC
|
F41
|
F: GGCTATGGAAGACTGGAGAGGG
|
551
|
[16]
|
R: GGGGTGACTGAGGTCATCCC
|
The Cytogenetics and Genomics Laboratory of the Faculty of Veterinary of the University of Murcia used the ileum and colon tissue samples for the detection of the virulence factor genes F4, F5, F41, F18, F6, LT, and STa by real-time PCR-based “Plus/Minus” assay (methods described on the Additional file). Primer sequences are described in Table 3. A commercial DNA extraction kit (DanaPure Spin Kit. Genedan S.L., Spain) was used to extract DNA from the ileum and colon according to the manufacturer's recommendation.
Table 3 – Characteristics of oligonucleotide sequences used for real-time PCR-based “Plus/Minus” assay to detect E. coli virulence factor genes.
PCR and samples
|
Virulence factor
|
Oligonucleotide sequence (5'-3')
|
Size of amplified product (bp)
|
Reference
|
Real-time PCR-based “Plus/Minus” assay, ileum and colon
|
F4
|
F: GGTTCAGTGAAAGTCAATGCATCT
|
70
|
[17]
|
R: CCCCGTCCGCAGAAGTAAC
|
F5
|
F: GCTATTAGTGGTCATGGCACTGTAG
|
80
|
[17]
|
R: TTTGTTTTCGCTAGGCAGTCATTA
|
F6
|
F: CCAAAGTATTCCACTGCAAGCA
|
72
|
[17]
|
R: GCCGTAACTCCACCGTTTGT
|
F18
|
F: TTGTGCTTCCTTGTCCAATAAAAC
|
82
|
[17]
|
R: CTCCCCCTTGATTAGCAAAACC
|
F41
|
F: CTGCTGATTGGACGGAAGGT
|
88
|
[17]
|
R: CCAGTCTTCCATAGCCATTTAACAG
|
LT
|
F: CCGGCAGAGGATGGTTACAG
|
73
|
[17]
|
R: GAATCCAGGGTTCTTCTCTCCAA
|
Sta
|
F: GCAAAATCCGTTTAACTAATCTCAAA
R:ACAGAAATAAAAATTGCCAACATTAGC
|
91
|
[18]
|
2.4.2. Identification of Porcine Enteric Disease Complex (PEDC) pathogens.
Refrigerated ileum and colon tissue samples were also used to identify PEDC pathogens. DNA was extracted using a commercial kit (DanaPure Spin Kit. Genedan S.L., Spain).
Afterwards, conventional PCR was performed using the technique described by Suh and Song (2005) [19] with a modification. Suh and Song (2005) [19] described a multiplex PCR for the simultaneous diagnosis of L. intracellularis, B. hyodisenteriae, and Salmonella spp. However, in order to increase the sensitivity of the technique, three simple PCRs for the diagnosis of each of the bacteria were performed.
If a positive result was found for any of the three pathogens, a real-time PCR would then be performed according to the methodology described in the Additional file. The Primers used are described in Table 4.
Table 4 – Characteristics of oligonucleotide sequences used for PCR assays to detect Porcine Enteric Disease Complex (PEDC) pathogens.
PCR and samples
|
Virulence factor
|
Oligonucleotide sequence (5'-3')
|
Size of amplified product (bp)
|
Reference
|
Conventional PCR, ileum and colon
|
L. intracellularis
|
F: GCAGCACTTGCAAACAATAAACT
|
210
|
[19]
|
R: TTCTCCTTTCTCATGTCCCATAA
|
B. hyodisenteriae
|
F: GCTGGAGATGATGCTTCTGG
|
403
|
[19]
|
R: GTCCAAGAGCTTGGCTGTTC
|
Salmonella spp.
|
F: TTGGTGTTTATGGGGTCGTT
|
298
|
[19]
|
R: GGGCATACCATCCAGAGAAA
|
Real-time PCR, ileum and colon
|
L intracellularis
|
F: GCGCGCGTAGGTGGTTATAT
|
98
|
[20]
|
R: GCCACCCTCTCCGATACTCA
|
2.5. Statistics
Data were included in an Excel database and analyzed by SPSS v. 22 15.0 (SPSS Inc., USA). The frequencies were analyzed by squared-Chi (χ2) with adjusted residues analysis and the quantity of DNA copies of the pathogens when real-time PCR was performed was analyzed by T-student. The correlations were calculated with Spearman's correlation test. A difference was considered significant when p<0.05.