Background
Methods
Sampling strategy and Salmonella isolation
Flock no. | No. of birds | Age/day | Clinical signs | Mortality, n (%) | Isolation results |
---|---|---|---|---|---|
1 | 10,000 | 1 | Pasty diarrhea, blindness, lameness and high mortality | 850 (8.5) |
S. Enteritidis |
2 | 10,000 | 2 | Inappetence and respiratory manifestation | 110 (1.1) | Negative |
3 | 15,000 | 3 | Pasty diarrhea, conjunctivitis, lowering in body weight and high mortalities | 975 (6.5) |
S. Enteritidis |
4 | 15,000 | 4 | Inappetence, ruffling feather and nervous signs | 360 (2.4) | Negative |
5 | 25,000 | 5 | Lowering body rate and respiratory signs | 550 (2.2) | Negative |
6 | 2000 | 7 | Pasty diarrhea, loss of appetite, ruffling feather and high mortalities | 190 (9.5) |
S. Typhimurium |
7 | 5000 | 7 | Decreased body weight, diarrhea, dehydration and high mortalities | 415 (8.3) |
S. Typhimurium |
8 | 20,000 | 4 | Decreased body weight | 500 (2.5) | Negative |
9 | 10,000 | 6 | Whitish diarrhea, high mortalities, and decreased body weight | 1160 (11.6) |
S. Typhimurium |
10 | 12,000 | 7 | Inappetence, diarrhea and lowering body weight | 540 (4.5) | Negative |
11 | 25,000 | 7 | Inability to move and nervous signs | 850 (3.4) | Negative |
12 | 30,000 | 5 | Diarrhea, drop in feed intake and high mortalities | 2610 (8.7) |
S. Typhimurium |
13 | 5000 | 4 | Respiratory signs and decreased body weight | 225 (4.5) | Negative |
14 | 30,000 | 3 | Inappetence, lowering growth rate | 840 (2.8) | Negative |
15 | 15,000 | 6 | Whitish diarrhea, conjunctivitis and decreased body weight | 945 (6.3) |
S. Typhimurium |
16 | 10,000 | 4 | Diarrhea and decrease in body weight and respiratory signs | 350 (3.5) | Negative |
17 | 10,000 | 5 | Inappetence, mortalities, lameness and diarrhea | 550 (5.5) |
S. Typhimurium |
18 | 20,000 | 6 | Decreased body weight and respiratory signs | 640 (3.2) | Negative |
19 | 12,000 | 5 | Diarrhea, blindness and high mortality | 648 (5.4) |
S. Typhimurium |
20 | 10,000 | 5 | Respiratory and nervous signs | 420 (4.2) | Negative |
21 | 15,000 | 33 | Inappetence and respiratory manifestation | 375 (2.5) |
S. Typhimurium |
22 | 20,000 | 32 | Decreased body weight | 280 (1.4) | Negative |
23 | 30,000 | 33 | Mortalities | 660 (2.2) | Negative |
24 | 25,000 | 29 | Nervous signs | 625 (2.5) | Negative |
25 | 15,000 | 34 | Decreased body weight | 345 (2.3) |
S. Typhimurium |
26 | 20,000 | 29 | Decreased body weight | 460 (2.3) | Negative |
27 | 30,000 | 30 | Decreased body weight | 780 (2.6) | Negative |
28 | 10,000 | 31 | Respiratory signs and high mortality | 330 (3.3) |
S. Typhimurium |
29 | 5000 | 28 | Respiratory signs and mortalities | 165 (3.3) | Negative |
30 | 15,000 | 33 | Inappetence and mortalities | 480 (3.2) | Negative |
31 | 20,000 | 33 | Respiratory signs and high mortality | 600 (3.0) |
S. Typhimurium |
32 | 20,000 | 32 | Inappetence and respiratory manifestation | 700 (3.5) |
S. Typhimurium |
33 | 10,000 | 28 | Mortalities | 200 (2.0) | Negative |
34 | 20,000 | 29 | Mortalities | 480 (2.4) | Non typable Salmonella (three isolates) |
35 | 10,000 | 33 | Respiratory signs and mortalities | 290 (2.9) |
S. Typhimurium |
36 | 5000 | 33 | Lower body weight and respiratory signs | 135 (2.7) | Negative |
37 | 15,000 | 32 | Nervous signs | 345 (2.3) | Negative |
38 | 20,000 | 33 | Inappetence and mortalities | 540 (2.7) | Negative |
39 | 10,000 | 31 | Respiratory signs and mortalities | 320 (3.2) |
S. Enteritidis |
40 | 5000 | 29 | Nervous manifestations and inappetence | 140 (2.8) | Negative |
41 | 25,000 | 35 | Opisthosomas and ruffled feather | 825 (3.3) | Negative |
Genomic DNA extraction and purification
Molecular biological identification and differentiation of Salmonella serovars
Gene | Oligonucleotide sequence (5′–3′) | Annealing (°C) | Amplicon size (bp) | Reference |
---|---|---|---|---|
invA-F | GCT GCG CGC GAA CGG CGA AG | 62 | 389 | [32] |
invA-R | TCC CGG CAG AGT TCC CAT T | |||
SdfI-F | TGTGTTTTATCTGATGCAAGAGG | 58 | 293 | [33] |
SdfI-R | CGTTCTTCTGGTACTTACGATGAC | |||
SdfII-F | GCGAATATCATTCAGGATAAC | 58 | 450 | [33] |
SdfII-R | GCATGTCATACCGTTGTGGA | |||
SdfIII-F | GCTGACTCACACAGGAAATCG | 58 | 350 | [33] |
SdfIII-R | TCTGATAAGACTGGGTTTCACT | |||
SefA-F | GCC GTA CAC GAG CTT ATA GA | 55 | 250 | [33] |
SefA-R | ACC TAC AGG GGC ACA ATA AC | |||
Sal fliC-F | CCCCGCTTACAGGTGGACTAC | 62 | 433 | [35] |
Sal fliC-R | AGCGGGTTTTCGGTGGTTGT | |||
SopE-F | ACA CAC TTT CCA CGA GGA AGC G | 55 | 398 | [36] |
SopE-R | GGA TGC CTT CTG ATG TTG ACT GG | |||
Typh-F | TTGTTCACTTTTTACCCCTGA A | 55 | 401 | [34] |
Typh-R | CCCTGACAGCCGTTAGATATT |
Determination and sequencing of class 1 integrons
Antimicrobial susceptibility testing
Antimicrobial agents | Conc. (µg) | Diameter of inhibition zone (mm) |
S. Typhimurium (58) |
S. Enteritidis (6) | Non typable (3) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
R | I | S | R | I | S | R | I | S | R | I | S | ||
Ampicillin | 10 | ≤13 | 14–16 | ≥17 | 58 (100%) | – | – | – | – | 6 (100%) | 3 (100%) | – | – |
Chloramphenicol | 30 | ≤12 | 13–17 | ≥18 | 58 (100%) | – | – | – | – | 6 (100%) | 3 (100%) | – | – |
Gentamicin | 10 | ≤12 | 13–15 | ≥16 | – | – | 58 (100%) | – | – | 6 (100%) | – | 3 (100%) | – |
Streptomycin | 25 | ≤11 | 12–14 | ≥15 | – | 6 (10.3%) | 52 (89.7%) | – | – | 6 (100%) | – | 3 (100%) | – |
Tetracycline | 30 | ≤14 | 15–18 | ≥19 | 58 (100%) | – | – | – | – | 6 (100%) | 3 (100%) | – | – |
Trimethoprim/sulphamethoxazole | 25 | ≤10 | 11–15 | ≥16 | 3 (5.2%) | – | 55 (94.8%) | – | – | 6 (100%) | – | 3 (100%) | – |
Gene | Primer | Nucleotide sequence (5–3) | Annealing (°C) | Amplicon size (bp) | Reference |
---|---|---|---|---|---|
aadA1 | F | TATCAGAGGTAGTTGGCGTCAT | 54 | 484 | [62] |
R | GTTCCATAGCGTTAAGGTTTCATT | ||||
aadA2 | F | TGTTGGTTACTGTGGCCGTA | 62 | 622 | [62] |
R | GATCTCGCCTTTCACAAAGC | ||||
aadB | F | GAGCGAAATCTGCCGCTCTGG | 61 | 319 | [62] |
R | CTGTTACAACGGACTGGCCGC | ||||
aacC | F | GGCGCGATCAACGAATTTATCCGA | 58 | 488 | [28] |
R | CCATTCGATGCCGAAGGAAACGAT | ||||
blaTEM | F | CATTTCCGTGTCGCCCTTAT | 55 | 793 | [62] |
R | TCCATAGTTGCCTGACTCCC | ||||
cat1 | F | CTT GTC GCC TTG CGT ATA AT | 53 | 508 | [27] |
R | ATC CCA ATG GCA TCG TAA AG | ||||
cat2 | F | CCGGATTGACCTGAATACCT | 56 | 572 | [62] |
R | TCACATACTGCATGATGAAC | ||||
dfrI | F | GTGAAACTATCACTAATGGTAGCT | 54 | 470 | [62] |
R | ACCCTTTTGCCAGATTTGGTAACT | ||||
floR | F | AACCCGCCCTCTGGATCAAGTCAA | 60 | 548 | [62] |
R | CAAATCACGGGCCACGCTGTATC | ||||
strA | F | AGCAGAGCGCGCCTTCGCTC | 59 | 684 | [62] |
R | CCAAAGCCCACTTCACCGAC | ||||
strB | F | ATCGTCAAGGGATTGAAACC | 49 | 509 | [63] |
R | GGATCGTAGAACATATTGGC | ||||
sul1 | F | TCACCGAGGACTCCTTCTTC | 60 | 316 | [62] |
R | AATATCGGGATAGAGCGCAG | ||||
sul2 | F | CGGTCCGGCATCCAGCAATCC | 64 | 441 | [62] |
R | CGAGAGCCACGACCGCGCC | ||||
sul3 | F | GAGCAAGATTTTTGGAATCG | 51 | 799 | [63] |
R | CATCTGCAGCTAACCTAGGGCTTGGA | ||||
tetA | F | GCTACATCCTGCTTGCCTTC | 55 | 210 | [64] |
R | CATAGATCGCCGTGAAGAGG | ||||
tetB | F | TTGGTTAGGGGCAAGTTTTG | 53 | 659 | [64] |
R | GTAATGGGCCAATAACACCG | ||||
tetC | F | CTTGAGAGCCTTCAACCCAG | 56 | 418 | [64] |
R | ATGGTCGTCATCTACCTGCC | ||||
intI | F | 5′GGCATCCAAGCAGCAAGC-3′ | 55 | 2000 | [65] |
R | AAGCAGACTTGACCTGAT |
Statistical analysis
Results
Clinical signs, mortality and incidence of Salmonella isolation from broiler flocks
Molecular biological identification of Salmonella serovars and public health significance
Organs | Liver | Intestine | Gallbladder | Total | |
---|---|---|---|---|---|
No. of collected samples | 205 | 205 | 205 | 615 | |
No. of isolates | |||||
S. Enteritidis | 2 (0.98%) | 1 (0.49%) | 3 (1.46%) | 6 (0.98%) | |
S. Typhimurium | 20 (9.76%) | 8 (3.9%) | 30 (14.63%) | 58 (9.43%) | |
Un-typable Salmonella
| 0 | 3 (1.46%) | 0 | 3 (0.49%) | |
Total | 22 (10.74%) | 12 (5.85%) | 33 (16.09%) | 67 (10.9%) |
Phenotypic and genotypic antimicrobial resistance
Resistance markers | Prevalence of resistance genes in screened Salmonella isolates | Antimicrobial agent | ||
---|---|---|---|---|
S. Typhimurium n = 58 |
S. Enteritidis n = 6 | Non-typable n = 3 | ||
BlaTEM
| 38 | 0 | 0 | Ampicillin |
tetA
| 49 | 3 | 3 | Tetracycline |
tetB
| 0 | 0 | 0 | Tetracycline |
tetC
| 56 | 2 | 0 | Tetracycline |
sul1
| 33 | 1 | 0 | Sulphamethoxazole |
sul2
| 0 | 0 | 0 | Sulphamethoxazole |
sul3
| 57 | 0 | 0 | Sulphamethoxazole |
aadA1
| 24 | 3 | 0 | Streptomycin |
aadA2
| 55 | 2 | 0 | Streptomycin |
strA
| 36 | 2 | 0 | Streptomycin |
strB
| 0 | 0 | 0 | Streptomycin |
aadB
| 0 | 0 | 0 | Gentamycin |
aacC
| 0 | 0 | 0 | Gentamycin |
cat1
| 58 | 2 | 1 | Chloramphenicol |
cat2
| 0 | 0 | 0 | Chloramphenicol |
floR
| 46 | 1 | 0 | Chloramphenicol |
dfrI
| 0 | 0 | 0 | Trimethoprim |
intI
| 3 | 0 | 0 | Class I integron |