**3. Key results**

#### **3.1** *Salmonella* **from feedlot cattle**

*Salmonella* was isolated from 58 out of 458 (12.7%) fecal samples tested (Tabe et al, 2010a, 2010b). All *Salmonella* belonged to the Typhimurium serotype and the majority 53/58 (91.4%) were Typhimurium *vars* Copenhagen. The rest (3/58, 5%) were reported as

National Veterinary Service Laboratories, Animal and Plant Health Inspection Services, U.S. Department of Agriculture, Ames, Iowa, for serotyping. PFGE assays on *Salmonella* cultures to investigate their genotypic relatedness were performed at the *E. coli* Reference Centre, Pennsylvania State University, University Park. The sample preparation, restriction digestion, electrophoresis, and gel staining for PFGE were accomplished following the CDCstandardized procedure as described (CDC, 2004) (http://www.cdc.gov/pulsenet/ protocols.htm). Restriction endonuclease *XbaI* (Roche Diagnostics Corporation, Indianapolis, IN) was used for restriction digestion of genomic DNA. The size standard used for all gels was *XbaI*-digested DNA from *Salmonella* Braenderup strain H9812 (American Type Culture Collection catalogue no. BAA-664), i.e. the universal size standard used by all PulseNet laboratories. Fingerprints were analyzed using BioNumerics software version 3.5 (Applied Maths, Austin, Texas). Strain relatedness was done based on previously recommended criteria (Gebreyes et al. 2006) using 'different bands' algorithm for clustering and the unweighted pair group for arithmetic means (UPGMA) tree-building approach with optimization of 1 and 0.5% position tolerance. Visual inspection of the patterns was

The bacterial DNA template preparation and the PCR conditions for the detection of class 1 and class 2 integrons were undertaken as previously described (Miko et al, 2005). The screening for the presence of class 1 and class 2 integrons was carried out using PCR with primers specific for the *intI1* ( and *intI2* (Goldstein et al, 2001)). The primer sequences used are shown in Table 2. Amplifications were performed in 10 μL of 5x Taq PCR Master Mix (Qiagen, Valencia, CA, USA), 2 pmol/L each primer, and 2 μg template DNA. Amplification specifications were as follows: 5 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 30 s at 72°C. PCR products were analyzed by gel electrophoresis with 2 % agarose

(bp)

*intI1* F: CCTCCCGCACGATGATC 280 55 *Kraft et al., 1986* 

*intI2* F: TATTGCTGGGATTAGGC 233 50 *Goldstein et al., 2001* 

Table 2. PCR primers and conditions used in Screening *Salmonella* isolates for presence of

*Salmonella* was isolated from 58 out of 458 (12.7%) fecal samples tested (Tabe et al, 2010a, 2010b). All *Salmonella* belonged to the Typhimurium serotype and the majority 53/58 (91.4%) were Typhimurium *vars* Copenhagen. The rest (3/58, 5%) were reported as

PCR Annealin g Temp (0C)

References

performed as a final step for analysis.

**2.6 PCR amplification of class 1 and 2 integrons** 

gels. All PCRs included DNA ladder, positive and negative controls.

Primers Sequence *a`* Size

R: TCCACGCATCGTCAGGC

R: ACGGCTACCCTCTGTTATC

**3.1** *Salmonella* **from feedlot cattle** 

**3. Key results** 

class 1 and class 2 integrons; *a* F, Forward; R, Reverse**.** 

one of the antibiotics (Table 3). All but two of the isolates were resistant to more than two of the antibiotics tested with 96.6% (56 of 58) of the isolates showing MDR antibiogram. All isolates tested were susceptible to amikacin, cefoxitin, cetriaxone, ciprofloxacin, gentamycin, gentamycin, nalidixic acid, and trimethoprim-sulfamethoxazole (Table 3). Almost all the isolates recovered from this study had a similar antimicrobial pattern. Regardless of sampling period (1, 2, or 3), 29 (3 Salmonella serovars Typhimurium and 26 Salmonella serovars Typhimurium var Copenhagen) were positive for class I integron (280 bp product) while only two of the isolates showed a 233-bp PCR product using primers *intI2* thus suggesting the presence of integron 2. These two isolates also had integron 1. Upon PFGE analysis, 9 distinguishable *Salmonella* genotypes were identified. For clarity, the genotypes were numbered I to IX with genotype V (28 of 58; 46.6%) being the most prevalent followed by type VII (15 of 58; 25.9 %) (Figure 1). Genotypes I, II, and III had the least prevalence (1 of 58; 1.7 % each). From the 58 isolates, types IV, V, VII, VIII, and IX (38 of 58; 65.5 %) isolated from the cattle at two sampling periods were observed at a similarity level of 100 %. Type V (28 of 58 isolates; 48.2 %) genotypes comprised of the most common isolates; of the 28 isolates from type V, 8 of 28 (28.6%), 18 of 28 (64.3%), and 2 of 28 (7.1%) were derived from sampling 1, 2 and 3 respectively. (Figure1). The 2 isolates which were positive for both *Int* 1 and 2 belonged to genotypes I and IV, respectively. Sampling time had a significant effect on the recovery of *Salmonella* (*P =* 0.004) while pen (*P* = 0.79) did not. All 58 *Salmonella* isolates which were grouped into two clusters (d and e) and five single isolates (a, b, c, f, and g) were observed at a similarity level of 80% (Figure 1).


Table 3. Number (%) of *Salmonella* isolates resistant/susceptible to various antimicrobials (N = 15)

Antimicrobial Drug Resistance and Molecular Characterization

resistant to Ampicillin and to Spectinomycin.

intermediate result to Sulphathiazole (Table 4).

**3.1.2** *Salmonella* **from dairy cattle** 

Resistant,

**3.1.3** *Salmonella* **from bison** 

of *Salmonella* Isolated from Domestic Animals, Humans and Meat Products 225

Penicillin, Sulphachlorophridazine, Sulphadimethoxime, Sulphathiazole, Tiamulin, Tilmicosin. Two isolates (both *Salmonella* Worthington), were susceptible to Ampicillin, whereas the other thirteen samples (all *Salmonella* Typhimurium (Copenhagen), were

In a study of Salmonella occurrence in dairy cattle (Khaitsa et al, 2004), 5 out of 30 cows (17%) tested positive for *Salmonella.* A sensitivity test to 20 antibiotics was performed on the 5 *Salmonella* isolates and the results were similar for all the 5 isolates except for only one isolate that was sensitive to Sulphachloropyridazine and Sulphadimethoxime and gave an

Antimicrobial All 5 Salmonella Isolates

Ampicillin R Apramycin S Ceftiofur S Chlortetracycline R Clindamycin R Enrofloxacin S Erythromycin R Florfenicol R Gentamicin S Neomycin S Oxytetracycline R Penicillin R Spectinomycin R Sulphachloropyridazine R (S)\* Sulphadimethoxime R (S)\* Sulphathiazole R *(I)\**  Tiamulin R Tilmicosin R

Trimethoprim/Sulphamethoxazole S Tylosin (Tartrate/Base) R S = Sensitive, I = Intermediate. \*These 3 antimicrobials are the only ones that gave a different result (sensitive or intermediate) to 1 of the 5 isolates; the other 4 isolates were all resistant to them).

Table 4. Antimicrobial Sensitivity Results to 5 *Salmonella* Isolates from dairy cattle. R =

The prevalence of *Salmonella* in the bison feces was 15% (3/20). The *Salmonella* isolates belonged to the serotypes *Salmonella* Typhimurium (Copenhagen) and *Salmonella* Worthington. In a panel of 20 antimicrobials, *Salmonella* Typhimurium (Copenhagen) was resistant to 13 of 20 antimicrobials (65% resistance), including macrolides (erythromycin, tilmicosin, tylosin), tetracyclines (chlortetracycline, oxytetracycline), chloramphenicol

For all other antimicrobials the results were the same for all 5 Salmonella isolates.

Percent similarity

Fig. 1. Dendogram generated from the *Xbal* patterns of the 58 *Salmonella* isolates using UPGMA clustering analysis with the BioNumerics software. A positive tolerance of 1.5 % was chosen.
