Prevalence and antimicrobial resistance patterns of Salmonella

0 downloads 0 Views 257KB Size Report
resistance patterns of Salmonella enterica isolated from preweaned calves and identify management risk factors associated with fecal shedding of S enterica.
05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1580

Prevalence and antimicrobial resistance patterns of Salmonella enterica in preweaned calves from dairies and calf ranches Anna Catharina B. Berge, DVM, MPVM, PhD; Dale A. Moore, DVM, MPVM, PhD; William M. Sischo, DVM, MPVM, PhD

Objective—To evaluate serovar and antimicrobial resistance patterns of Salmonella enterica isolated from preweaned calves and identify management risk factors associated with fecal shedding of S enterica. Sample Population—Cohorts of 10 to 15 preweaned calves (1 to 84 days of age) on 26 dairies and 7 calf ranches and cross-sectional samples of preweaned calves on smaller farms. Procedures—Calves were evaluated every 2 weeks during a 6-week period. Salmonella isolates obtained from rectal fecal swabs underwent antimicrobial susceptibility testing against 12 antimicrobials. Cluster analysis enabled description of antimicrobial susceptibility patterns. Calf, cohort, and farm risk factors associated with both the prevalence of S enterica and multiple-antimicrobial–resistant S enterica in preweaned calves were identified with repeated-measure logistic regression models. Results—Salmonella enterica was detected on > 50% of farms and in 7.5% of 3,686 fecal samples. Many isolates (33%) were resistant to multiple antimicrobials. Shedding of Salmonella spp was negatively associated with increasing calf age, herds being closed to incoming cattle, and antimicrobial supplementation of milk replacer; prophylactic antimicrobial treatment in day-old calves increased shedding. No association between farm management and presence of multiple-antimicrobial–resistant S enterica or between calving management and presence of S enterica in calves ≤ 1 week old was detected. Conclusions and Clinical Relevance—In preweaned calves, the most important factors associated with decreased likelihood of fecal shedding of S enterica were the use of antimicrobial-supplemented milk replacer and maintenance of a closed herd. Infection with multipleantimicrobial–resistant S enterica was not associated with antimicrobial administration. (Am J Vet Res 2006;67:1580–1588)

S

almonella enterica is one of many enteric disease–causing agents in humans and other animals. Salmonella spp are zoonotic and often foodborne Received October 30, 2005. Accepted January 4, 2006. From the Department of Population Health and Reproduction, Veterinary Medicine Teaching and Research Center, University of California, Tulare, CA 93274. Supported by National Integrated Food Safety Initiative-Cooperative State Research, Education, and Extension Service (Grant No. 0051110-9721). Address correspondence to Dr. Berge. 1580

ARC GEE

ABBREVIATIONS Antimicrobial resistance cluster Generalized estimating equation

bacteria; major reservoirs for human infection are believed to be livestock and poultry.1 Salmonella spp are frequently isolated from the environment and livestock of dairy farms and calf ranches. Although Salmonella isolates can be obtained from nonclinically affected animals, these organisms are also associated with considerable illness and death among bovids.2-4 Typically, Salmonella isolates from nonclinically affected animals are susceptible to antimicrobials, whereas the isolates from clinically affected animals have higher overall levels of antimicrobial resistance.5,6 There are conflicting data regarding the influence that calf and dairy farm management practices, specifically the use of antimicrobials, have on fecal shedding of Salmonella spp and the development of antimicrobial resistance in those organisms. Results of 1 study7 of herd-level risk factors for the recovery of Salmonella spp from the environments of free-stall housing occupied by adult dairy cows indicated that there was no relationship between production or management factors and the prevalence of Salmonella spp. Other studies8,9 in adult dairy cattle have revealed an association of increasing herd size with increased fecal shedding and prevalence of Salmonella spp. In a national US study9 that was focused on heifers, 1,063 farms were surveyed; Salmonella spp were detected in 145 of 6,861 (2.1%) fecal samples obtained from preweaned heifers and 79 of 1,063 (7.4%) dairies from which 1 or more Salmonella-positive fecal samples had been collected. Feeding hay or antimicrobial–supplemented milk replacer to calves during the preweaning period was associated with lower risk for fecal shedding of Salmonella spp, compared with calves that were fed nonsupplemented milk or no hay. In another study,10 fecal samples were collected from calves every 2 months for 1 year on 129 conventional and organic dairy farms in 4 states; 176 of 4,673 (3.8%) fecal samples from calves and 40 (31%) farms yielded positive results for Salmonella spp. Herd-level factors associated with isolation of Salmonella spp from calves included feeding milk replacer without antimicrobial supplementation, use of the maternity pen as a hospital area for sick cows, and high prevalence of Salmonella spp in the cows. Antimicrobial use in calves and the risk for fecal shedding of Salmonella spp have been investigated furAJVR, Vol 67, No. 9, September 2006

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1581

ther. Hinton et al11 found that calves fed milk supplemented with furazolidone had peak fecal shedding of Salmonella spp from 2 to 3 weeks of age and that shedding rates decreased after 5 weeks of age. During the second week of life, diseased calves treated with antimicrobials were more likely to shed Salmonella spp than untreated calves. However, the level of Salmonella shedding in those calves was lower than that determined by those investigators in a previous study12 in which in-feed administration of antimicrobials to calves was used. The authors concluded that exposure to antimicrobials in feed decreased the probability of recovering Salmonella spp from fecal samples, but calves with clinical disease that were treated with antimicrobials were more likely to shed Salmonella spp than healthy, untreated calves. This latter effect may not be related to exposure to antimicrobials, but rather to the disease state. Most of these studies were crosssectional in design, and the findings are difficult to interpret when evaluating management practices as causal factors for fecal shedding of Salmonella spp. In addition, none of those studies assessed the impact of management practices and the use of antimicrobials on the recovery of multiple-antimicrobial–resistant Salmonella serovars. The objective of the study reported here was to evaluate serovar and antimicrobial resistance patterns of S enterica isolated from preweaned calves and identify management risk factors associated with fecal shedding of Salmonella spp. In addition, calf-level risk factors associated with the presence of multipleantimicrobial–resistant Salmonella spp in fecal samples obtained longitudinally from preweaned calves were investigated. Materials and Methods Farm enrollment and sampling strategy—Thirty-three farm operations that reared calves for veal, fed beef, or heifer replacements were selected from a convenience sample identified by University of California Cooperative Extension farm advisors and private veterinarians. The farms were located throughout California and included animals from the 4 major milk production areas in the state (North Coast, North Valley, South Valley, and Southern California). The farms represented dairy and calf ranch operations. Farm management personnel responded to a questionnaire (30-minute completion time) that was administered by an investigator (ACB) and gave permission for sample collection from calves. The questionnaire addressed farm type and size, biosecurity management (animal introduction onto the farm), antimicrobial treatments of cows, calving and colostrum management, type of calf housing and cleaning of housing, calf feeding management, and the use of prophylactic and therapeutic calf treatments. Farm operators maintained records of all antimicrobial treatments administered to calves in the trial cohorts. On farms where treatment records were not in compliance with study requirements, a temporary recording system was established at the first farm visit. For each farm, 1 to 3 cohorts were enrolled at approximately 6-month intervals (Table 1). At 5 farms, only 1 cohort was enrolled because of sampling logistics or changes in farm management. Ten to 15 newborn calves, or those newly arrived at a farm, were enrolled as a cohort. Each calf was individually identified. For smaller dairies, as many as 15 calves that were < 4 weeks of age were enrolled on the first visit; on subsequent visits to these smaller operations, calves AJVR, Vol 67, No. 9, September 2006

born between visits were enrolled. Overall, 1,288 calves were included in the study. Fecal samples were collected from each cohort on 4 occasions at 2-week intervals. Bacteriologic culture and antimicrobial resistance typing—Rectal fecal samples were obtained with sterile cottontipped transportation swabs.a Results of a prior pilot study by one of the authors indicated that Salmonella isolates on the swabs remained viable and bacterial counts were unchanged for as long as 2 weeks at 20oC. Study investigators collected the samples and transported them directly to the laboratory in cool, insulated containers within 24 hours of sampling. Fecal swabs were initially incubated for 18 to 24 hours at 37oC in 10 mL of tetrathionate broth.b Approximately 0.1 mL of the tetrathionate broth was then transferred to xylose lysine desoxycholate agarc and brilliant green agar platesc that were streaked for isolation and incubated for 18 to 24 hours at 37oC. Plates were evaluated for growth of Salmonella organisms, and 1 putative Salmonella colony from each Salmonella-positive plate was streaked onto blood agar and incubated 18 to 24 hours at 37oC. For biochemical confirmation, well-isolated colonies were inoculated to triple sugar ironc slants and urea agar slants and incubated at 37oC for 24 hours. Urease-negative, dextrose-fermenting, H2S-producing colonies were presumed to be Salmonella spp and transferred to blood agar plates for serotyping. Salmonella isolates were serogrouped by use of a commercial polyvalent A-I and Vi anti-sera as well as individual serogroups B, C1, C2, C3, D, and E.d One isolate/serogroup identified in each fecal swab was sent to the California Animal Health and Food Safety Laboratory for serovar determination. Salmonella isolates were kept in tryptic soy brothb with 10% glycerol (vol/vol) at –80oC. Antibiograms—Antimicrobial disk susceptibility tests were performed as recommended by the Clinical and Laboratory Standards Institute (previously named the National Committee for Clinical Laboratory Standards [NCCLS]) and as previously described.13-15 Twelve antimicrobials from the panel used by the National Antimicrobial Resistance Monitoring System were selected for the tests.5 This selection represented the major antimicrobial classes in historical and present use, and the drugs were chosen because of their importance in animal and human health. The 12 antimicrobials and their concentrations were the following: amikacin, 30 μg; amoxicillin-clavulanic acid, 20 μg/10 μg; ampicillin, 10 μg; cephalothin, 30 μg; ceftiofur, 30 μg; chloramphenicol, 30 μg; gentamicin, 10 μg; nalidixic acid, 30 μg; streptomycin, 10 μg; sulfisoxazole, 250 μg; tetracycline, 30 μg; and sulfamethoxazole-trimethoprim 23.75 μg/1.25 μg.b For each batch of isolates tested, 2 quality-control strains were included in the assay set: Escherichia coli ATCC 25922e from the American Type Culture Collection and S enterica serovar Typhimurium (bovine clinical isolate from the Veterinary Medicine Teaching and Research Center, University of California). Antimicrobial susceptibility patterns—Data from the antibiograms for each isolate consisted of a series of inhibition zones for each of the 12 antimicrobials. Zone sizes ranged from 6 mm in diameter (corresponding to the size of an antimicrobial disk) to 31 mm. For each antimicrobial, the distribution of zone sizes for the isolate set was determined.f Isolates were grouped into ARCs on the basis of patterns of similarity in the zone sizes to the 12 antimicrobials.g Clustering was identified by use of the squared Euclidean distance as a dissimilarity measure and Ward’s minimum variance clustering algorithm.16,17 For each cluster, the mean antimicrobial zone size and SD were calculated for each of the 12 antimicrobials.18 Antimicrobial resistance clusters were 1581

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1582

ordered on the increasing sum of the mean zone sizes, which roughly corresponded to increasing levels of multiple resistance. The clustering algorithm performs most efficiently when the distribution of zone sizes is bimodal; therefore, the distribution of the zone sizes was tested prior to clustering. All antimicrobial inhibition zone sizes had a bimodal distribution and were included in the cluster analysis procedure. For illustrative and descriptive purposes, the mean zone sizes for each antimicrobial within an ARC were described as susceptible or resistant according to the Clinical and Laboratory Standards Institute guidelines for human and animal E coli.15 Isolates in ARCs with resistance to 2 or more antimicrobials were described as multiple-antimicrobial–resistant isolates. Statistical modeling—To analyze the relationship between prevalence of Salmonella spp and risk factors, GEE logistic regression was used to account for the sampling of multiple cohorts on a farm. The nonindependence of isolates was modeled as a repeated measure of samples in a cohort nested by farm. Three separate models were used to describe the risk factors for fecal shedding of Salmonella spp in preweaned calves, risk factors for fecal shedding of multiple-antimicrobial–resistant Salmonella spp in preweaned calves, and calving-associated risk factors on dairy farms for fecal shedding of Salmonella spp in calves ≤ 7 days of age.

The modeling approach has been previously described.18 Briefly, the distribution of the predictive variables, conditional on Salmonella shedding status, was initially assessed by use of bivariate analyses. Prior to developing the GEE model, the set of variables identified in the bivariate analyses (χ2 test, P value ≤ 0.1) was evaluated in a population-averaged maximum-likelihood logistic regression model.19,h A stepwise variable selection procedure was used to build models. The procedure was based on a score χ2 test; a value of P ≤ 0.30 was required for entry into the model, and a value of P ≤ 0.10 was required for retention. Two-way interaction effects between variables included in this initial selection process were also tested. The variables and interaction terms retained in the population-averaged model were introduced to a GEE logistic regression model with a repeated measure of samples in a cohort nested within farm. An independent correlation structure was used for the repeated-measures component.20,21,i A value of P ≤ 0.10 was required for a risk factor to be retained in the final model.

Results Salmonella isolates were recovered from 278 of 3,686 (7.5%) fecal samples collected from preweaned calves, 18 of 33 (55%) farms, and 27 of 62 (44%) cohorts (Table 1). The fecal prevalence of Salmonella isolates was 8.3% (215/2,597) and 5.8% (63/1,089) from dairy calves and

Table 1—Characteristics of study farms and enrolled calf cohorts included in a study of prevalence and risk factors for fecal shedding of Salmonella enterica in preweaned calves in California. Cohort 1

Cohort 2

No. of Salmonellapositive samples

Cohort 3

No. of samples collected

No. of Salmonellapositive samples

No. of samples collected

No. of Salmonella positive samples

Milk shed

Farm type

Farm subtype

No. of samples collected

NC

Dairy Dairy Dairy Dairy Dairy Dairy Dairy Dairy Dairy

Organic Conventional Conventional Conventional Conventional Conventional Conventional Organic Organic

23 26 42 56 53 41 44 22 21

0 0 0 0 0 0 0 0 0

51 61 47 19 57 53 58 ND ND

0 0 0 0 0 0 0 — —

ND ND ND ND ND ND ND ND ND

— — — — — — — — —

NV

Dairy Calf ranch Dairy Dairy Dairy Dairy

Conventional Heifer Conventional Conventional Conventional Conventional

37 34 73 41 47 41

0 0 19 0 2 0

32 ND 51 37 39 40

0 — 0 0 0 2

ND ND ND ND ND ND

— — — — — —

SV

Dairy Dairy Dairy Dairy Dairy Calf ranch Calf ranch Calf ranch Calf ranch Dairy Dairy Dairy Dairy Calf ranch

Conventional Conventional Conventional Conventional Organic Beef Veal, heifer Beef Heifer Conventional Conventional Conventional Conventional Veal, heifer

58 38 41 56 65 58 129 105 48 115 67 111 117 106

15 0 0 2 0 0 6 0 1 29 4 20 22 6

50 41 57 55 41 53 52 121 ND ND ND 117 113 59

0 0 0 13 2 2 1 9 — — — 24 18 1

ND ND ND ND ND ND ND 141 ND ND ND ND ND 57

— — — — — — — 4 — — — — — 0

SO

Dairy Calf ranch Dairy Dairy

Conventional Heifer Conventional Conventional

55 55 52 50 1,927

0 31 26 0 183

66 69 55 63 1,557

0 2 7 10 91

ND ND ND ND 198

— — — — 4

Total

NC = North Coast. NV = North Valley. SV = South Valley. SO = Southern California. ND = Not done. — = Not applicable.

1582

AJVR, Vol 67, No. 9, September 2006

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1583

calf ranch calves, respectively. Salmonella Montevideo was the most common serovar recovered and accounted for 106 of 278 (38%) isolates. Other Salmonella serovars detected on several farms included the following: Give, Infantis, Meleagridis, Muenster, Newport, and Typhimurium. Salmonella serovar Minnesota was isolated only from calves on 3 farms in Southern California. The highest recovery of Salmonella organisms was in samples from 1-day-old calves, with declining prevalence as the calves aged (Figure 1). Multiple-antimicrobial–resistant Salmonella isolates were recovered from 92 of the 3,686 (2.5%) fecal samples. There were no detectable shifts in prevalence of multiple-antimicrobial–resistant Salmonella spp in calves with increasing age.

Fourteen ARCs described the patterns of resistance observed in the Salmonella isolates. Clusters were ordered from pan-susceptible (cluster A) to highly resistant (cluster N) on the basis of the sum of the mean zone sizes associated with the 12 antimicrobials (Table 2). Of the isolates, 169 of 278 (61%) were placed in ARC A and 92 of 278 (33%) were classified as multiple-antimicrobial–resistant organisms (ARCs D through N). The most common Salmonella serovar, Montevideo, was detected only in the most susceptible ARCs (A through C). Serovars with high levels of multiple-antimicrobial resistance (ie, resistant to 6 or more antimicrobials) included Dublin, Infantis, Minnesota, Muenster, Newport, Reading, and Typhimurium (Table 3). Most dairy farms and all calf ranches purchased animals from other sources (Table 4). Eight dairy farms were closed herds with no reported introduction of bovids in the preceding year. Preweaned calves were generally raised in individual, wooden hutches. Most farms removed manure from the hutches by either scraping or flushing, but only after a calf was weaned. On dairy farms, calves were typically fed fresh unpasteurized milk, whereas on calf ranches, calves were typically fed milk replacer (Table 5). Nonsaleable milk (ie, milk not allowed for human consumption) was fed to calves on dairy farms and 2 calf ranches. Pasteurization of fresh and nonsaleable milk was practiced on several dairy farms and all calf ranches that fed nonsaleable milk. All calf ranches used antimicrobials in the feed, whereas only 5 of 26 dairies used antimicrobials in Figure 1—Prevalence of Salmonella enterica in fecal samples collected from preweaned dairy-source calves on 33 farms in California. Overall, 3,685 fecal samples were collect- the feed. Approximately half of the dairies started feeding calves with bottles and ed from 1,288 calves during their first 12 weeks of life. Values are mean ± SD. Table 2—Characteristics of ARCs in a study of prevalence and risk factors for fecal shedding of Salmonella enterica in preweaned calves in California. No. of antimicrobials to which the isolates were ARCs resistant AMP A 0 B 1 C 1 D† 2 E† 3 F† 4 G† 5 H† 6 I† 8 J† 9 K† 9 L† 10 M† 11 N† 12 Total No. of isolates

23 24 23 23 6* 6* 6* 6* 6* 6* 6* 6* 6* 6*

Antimicrobial AMC

CEF

CFF

STR

GEN

AMI

SULF

SXT

TET

CHL

NAL

25 25 25 26 7* 19 17 20 7* 8* 6* 7* 8* 8*

23 24 23 24 6* 19 19 16 6* 6* 6* 6* 6* 6*

25 26 25 25 27 25 24 24 14* 13* 13* 15* 11* 12*

14 8* 12 6* 12 6* 6* 6* 6* 6* 6* 6* 6* 6*

22 22 22 18 21 22 19 6* 21 6* 21 8* 6* 6*

22 22 21 22 23 23 24 23 22 23 23 22 6* 6*

22 20 6* 6* 19 6* 6* 6* 6* 6* 6* 6* 6* 6*

27 27 23 23 24 22 19 6* 20 22 6* 7* 6* 7*

21 21 20 19 19 9* 8* 6* 6* 6* 6* 6* 6* 6*

24 25 25 21 20 24 8* 16 6* 6* 6* 6* 6* 6*

21 21 21 22 23 21 21 17 21 22 23 22 22 7*

Frequency of Salmonella isolates belonging to susceptibility clusters 169 12 5 9 2 6 4 1 5 2 21 3 15 24 278

*Mean zone sizes within a cluster were considered resistant to that antimicrobial; resistance classification was performed according to the Clinical and Laboratory Standards Institute guidelines for Escherichia coli isolated from humans. †Clusters classified as multiple-antimicrobial–resistant clusters. AMP = Ampicillin. AMC = Amoxicillin-clavulanic acid. CEF = Cephalothin. CFF = Ceftiofur. STR = Streptomycin. GEN = Gentamicin. AMI = Amikacin. SULF = Sulfisoxazole. SXT = Sulfamethoxazole-trimethoprim. TET = Tetracycline. CHL = Chloramphenicol. NAL = Nalidixic acid.

AJVR, Vol 67, No. 9, September 2006

1583

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1584

Table 3—Frequency of Salmonella enterica serovars and their ARC distribution in a study of the prevalence and risk factors for fecal shedding of Salmonella spp in preweaned calves in California. ARCs

Salmonella serovars

No. of isolates A*

B

C

D

E

F

G

H

I

J

K

L

M

N†

Agona Anatum Cerro Dublin Give

2 7 5 8 15

1 7 5 1 15

— — — 4 —

— — — — —

— — — — —

— — — — —

— — — — —

1 — — 1 —

— — — 1 —

— — — — —

— — — — —

— — — — —

— — — 1 —

— — — — —

— — — — —

Infantis Kentucky Lille Livingstone Mbandaka

13 7 1 6 7

— 1 1 3 5

— 5 — — 2

— — — 3 —

— — — — —

— 1 — — —

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

— — — — —

13 — — — —

— — — — —

Meleagridis Minnesota Montevideo Muenster Newington

19 25 106 9 1

18 — 103 7 1

— — 1 — —

— — 2 — —

— — — — —

1 — — — —

— — — — —

— — — — —

— — — — —

— 1 — — —

— — — — —

— — — — —

— — — — —

— — — 2 —

— 24 — — —

21 4 1 21

— — 1 —

— — — —

— — — —

— — — 9

— — — —

— — — 6

— — — 2

— — — —

2 2 — —

— 2 — —

17 — — 4

2 — — —

— — — —

— — — —

278

169

12

5

9

2

6

4

1

5

2

21

3

15

24

Newport Reading Schwarzengrund Typhimurium Total No. of isolates

*Pan-susceptible to 12 antimicrobials. †Pan-resistant to 12 antimicrobials. See Table 1 for remainder of key.

then switched to buckets, whereas calf ranches fed calves with bottles throughout the preweaning period. The final GEE logistic regression model for whether preweaned calves would shed Salmonella ssp in their feces contained factors for calf age, administration of antimicrobials in feed, biosecurity, and administration of antimicrobials within 24 hours after birth (Table 6). The odds that preweaned calves would shed Salmonella spp in their feces decreased significantly as age increased; calves fed antimicrobials in milk replacer were less likely to shed Salmonella spp in their feces than were calves that did not receive antimicrobials in milk replacer. Calves receiving prophylactic antimicrobials on the first day of life (day 1 treatments) were more likely to shed Salmonella spp in their feces at some time in the sampling period, compared with untreated calves. Antimicrobials used for these day 1 treatments included ceftiofur, penicillin, and gentamicin. Furthermore, calves from open herds were significantly more likely to shed Salmonella spp in feces than were calves from closed herds; this effect was consistent whether herds received purchased calves, heifers, or adult replacements. No management or feeding risk factors were associated with fecal shedding of multiple-antimicrobial–resistant Salmonella spp from preweaned calves. Calving management factors and colostrum regimen were not associated with Salmonella isolation in calves ≤ 1 week of age on dairy farms. Discussion In the present study, the prevalence of S enterica in fecal samples collected from preweaned calves was 7.5%. In addition, 55% of the dairies and calf ranches 1584

yielded Salmonella spp via bacteriologic culture at 1 or more of the sample collections. Many Salmonella serovars (Agona, Anatum, Dublin, Kentucky, Montevideo, Muenster, Newport, and Typhimurium) detected in our study have been identified in a national investigation5 of the prevalence of Salmonella spp in bovids. Most of the Salmonella isolates were pan-susceptible to the antimicrobials tested. Consistent with other reported data,5 Salmonella Montevideo was the most common pan-susceptible serovar detected in the present study. Ten of the 19 serovars included multiple-antimicrobial–resistant isolates. Multiple-antimicrobial–resistant Salmonella Minnesota has not been previously described and was detected in only 1 geographic region; one of the authors is aware that it has since been isolated from cattle in other regions of California. The focus of this research was to identify risk factors for fecal shedding of Salmonella spp in preweaned calves. The sampling and microbiologic methods (based on use of a fecal swab and a 1-step enrichment process) could identify animals that were shedding > 20 Salmonella CFUs/g of feces. Although it is likely that the sensitivity of the bacteriologic culture of Salmonella spp from feces can be improved with increased size of the fecal sample, we believe that many animals that are shedding low numbers of Salmonella organisms are not actively infected but that this low-level shedding reflects environmental contamination. Certain risk factors, including maintenance of a closed herd, exposure of calves to antimicrobial-supplemented milk replacer, antimicrobial prophylactic treatment of day-old calves, and calf age, were associAJVR, Vol 67, No. 9, September 2006

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1585

ated with fecal shedding of Salmonella spp by calves. The strongest risk factor was associated with biosecurity; farms that received animals from other sources

were 35 times as likely to have calves that were shedding Salmonella spp in feces than herds that did not receive such animals. This effect was consistent

Table 4—Categorization of 26 dairies and 7 calf ranches in which preweaned calves were reared in California by management and housing practices and number of fecal samples that were positive for S enterica. Dairies

Risk factor*

No. of farms

No. of fecal samples

Calf ranches No. of Salmonellapositive fecal No. of samples (%) farms

No. of SalmonellaNo. of positive fecal fecal samples samples (%)

Bovids purchased within last year Weekly (biosecurity open) Replacement heifers (biosecurity open) Breeding stock (biosecurity open) None (biosecurity closed) Calf housing Individual hutches Group housing Both

0 14 4 8

— 1,613 390 594

— 188 (11.7) 25 (6.4) 2 (0.3)

7 0 0 0

1,089 — — —

63 (5.8) — — —

24 2 1

2,395 116 86

211 (8.8) 2 (1.7) 2 (2.3)

7 0 0

1,089 — — —

63 (5.8) — —

Hutch type Wood Plastic Steel and wire

15 6 4

1,582 535 364

177 (11.2) 34 (6.4) 2 (0.5)

7 0 0

1,089 — —

63 (5.8) — —

11 19

906 1,691

133 (14.7) 82 (4.8)

6 1

1,089 —

63 (5.8) —

20 7

1,928 669

185 (9.6) 30 (4.5)

7 0

1,089 —

63 (5.8) —

9 9 7

800 789 892

59 (7.4) 31 (3.9) 123 (13.8)

0 1 6

— 181 908

— 7 (3.9) 56 (6.2)

9 7 6 2 3

705 958 582 180 222

31 (4.4) 134 (14.0) 48 (8.2) 0 (0.0) 2 (0.9)

2 4 1 0 0

292 673 124 — —

9 (3.1) 21 (3.1) 33 (26.6) — —

14 10 3

1,335 1,036 226

144 (10.8) 69 (6.7) 2 (0.9)

5 2 0

797 292 —

54 (6.8) 9 (3.1) —

19 2 5

1,828 172 597

163 (8.9) 2 (1.2) 50 (8.4)

4 3 0

429 660 —

41 (9.6) 22 (3.3) —

8 16

904 1,693

90 (10.0) 125 (7.4)

3 4

451 638

14 (3.1) 49 (7.7)

9 16

1,189 1,408

106 (8.9) 109 (7.7)

5 2

929 160

60 (6.5) 3 (1.9)

18 7

1,802 795

122 (6.8) 93 (11.7)

5 1

305 784

40 (13.1) 23 (2.9)

13 13

1,276 1,321

171 (13.4) 44 (3.3)

7 0

1,089 —

63 (5.8) —

8 18

1,067 1,530

162 (15.2) 53 (3.5)

5 2

797 292

54 (6.8) 9 (3.1)

17 3 6

1,669 373 555

160 (9.6) 40 (10.7) 15 (2.7)

6 1 0

908 181 —

56 (6.2) 7 (3.9) —

Elevation from ground Yes No Bedding or floor material Organic Inorganic Separation between hutches 1–3 ft (0.3−0.9 m) None Triplet hutch Ground surface around calves Concrete Soil Sand Rocks Grass Manure removal method Scraping Flushing Hutch movement Manure removal frequency Between cohorts Weekly Daily Mechanical cleaning Yes No High-pressure washing Yes No Hutch disinfectant used None Yes Liming of hutches Yes No Sun drying of hutches between cohorts Yes No Period that hutches are empty between calf cohorts 1–7 days 8–89 days ⱖ 90 days

*For a given risk factor, number of dairy farms exceeds 26 when there was a management change in that factor on 1 or more farms between trial cohorts. See Table 1 for remainder of key.

AJVR, Vol 67, No. 9, September 2006

1585

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1586

Table 5—Categorization of 26 dairies and 7 calf ranches in which preweaned calves were reared in California by calf feeding and antimicrobial use practices and number of fecal samples that were positive for S enterica. Dairies

Calf ranches

No. of SalmonellaNo. of positive fecal fecal samples samples (%)

No. of SalmonellaNo. of positive No. of fecal fecal farms samples samples (%)

Risk factor*

No. of farms

Fresh milk Not fed Pasteurized Unpasteurized

4 3 19

549 299 1,749

65 (11.8) 19 (6.4) 131 (7.5)

7 0 0

1,089 — —

63 (5.8) — —

Hospital milk† Not fed Pasteurized Unpasteurized

6 6 14

561 618 1,418

35 (6.2) 44 (7.1) 136 (9.6)

5 3 0

877 212 —

59 (6.7) 4 (1.9) —

Milk replacer Yes No

10 16

1,153 1,444

155 (13.4) 60 (4.2)

7 0

1,089 —

63 (5.8) —

No. of daily feedings 2 3

26 0

2,597 —

215 (8.3) —

5 2

795 294

50 (6.3) 13 (4.4)

Feeding by bottle Yes No

25 1

2,486 111

200 (8.0) 15 (13.5)

7 0

1,089 —

63 (5.8) —

Feeding by bucket Yes No

15 11

1,343 1,254

63 (4.7) 152 (12.1)

1 6

165 924

7 (4.2) 56 (6.1)

Medicated feed Yes No

5 22

465 2,132

23 (4.9) 192 (9.0)

7 0

1,089 —

63 (5.8) —

In-feed antimicrobial administration None In milk In grain

22 3 2

2,132 266 199

192 (9.0) 23 (8.6) 0 (0.0)

0 6 1

— 965 124

— 30 (3.1) 33 (26.6)

Introduction of grain into diet 0–4 days 5–7 days ⬎ 7 days

18 7 1

1,866 618 113

169 (9.1) 36 (5.8) 10 (8.8)

6 1 0

743 111 —

49 (6.6) 2 (1.8) —

Use of coccidiostat No Yes Milk Grain Both

9 17 3 13 1

877 1,720 438 1,195 87

83 (9.5) 132 (7.7) 59 (13.5) 73 (6.1) 0 (0.0)

2 5 1 3 1

408 681 116 513 52

46 (11.3) 17 (2.5) 1 (0.9) 15 (2.9) 1 (1.9)

Administration of antimicrobials prophylactically within first 24 hours after birth Yes No

0 26

— 2,597

— 215 (8.3)

3 6

295 794

39 (13.2) 24 (3.0)

Individual antimicrobial treatment of calves within 14 days of sample collection Yes No

— —

251 2,346

21 (8.4) 194 (8.3)

— —

195 894

12 (6.2) 51 (5.7)

*For a given risk factor, number of dairy farms exceeds 26 and number of calf ranches exceeds 7 when there was a management change in that factor on 1 or more farms or ranches between trial cohorts. † ??? See Table 1 for remainder of key.

whether herds purchased calves, heifers, or adult replacements. This may be attributable to the introduction and dissemination of Salmonella spp into farms via animals. Closed herds may also have other biosecurity measures in place that prevent the intro1586

duction and dissemination of Salmonella spp, such as restrictions on incoming visitors, vehicles, or feed. Calves that received antimicrobial-supplemented milk replacer throughout the preweaning period were less likely to shed Salmonella spp in feces, compared AJVR, Vol 67, No. 9, September 2006

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1587

Table 6—Odds ratios derived from a GEE multilevel logistic regression model for the risk of fecal shedding of S enterica in preweaned calves on 26 dairies and 7 calf ranches in California. Odds ratio

90% confidence interval

P value

Variable

Estimate

Calf age (d) Continuous

–0.04

0.97

0.95–0.98

⬍ 0.01

Antimicrobials in feed Grain Milk No

–0.10 –1.47 Reference

0.90 0.23 —

0.35–5.60 0.09–0.59 —

0.87 0.01 —

Biosecurity Open herd Closed herd

3.57 Reference

35.52 —

7.11–178.50 —

Antimicrobials administered during first 24 hours after birth Yes No

1.21 Reference

3.35 —

1.09–10.33 —

⬍ 0.01 —

0.08 —

A value of P ⱕ 0.10 was considered significant. See Table 1 for key.

with calves that received no antimicrobials in milk or milk replacer or only antimicrobials in the starter grain. The lower recovery of Salmonella isolates from feces of calves exposed to antimicrobial-supplemented milk replacer during the preweaning period was likely attributable to suppression of pan-susceptible Salmonella serovars. In other studies,9,10 a similar association between the use of antimicrobial-supplemented milk replacer and decreased odds of fecal shedding of Salmonella spp by preweaned calves has been determined. In contrast to the protection that prolonged use of prophylactic antimicrobials in the milk or milk replacer appeared to have on fecal shedding of Salmonella spp, a single prophylactic antimicrobial treatment of calves on their first day of life was associated with an increased recovery of Salmonella spp. Antimicrobial therapy can disrupt the normal, commensal enteric flora.22 This disruption may allow either colonization or overgrowth of nondominant organisms (such as Salmonella spp) and subsequent shedding. The class of antimicrobial and the singledose treatment may have different selective effects on the gut flora and result in a markedly different response on Salmonella shedding for the day 1 treatment versus in-feed antimicrobials. It is also possible that the day 1 prophylactic use may be a response to a farm history of Salmonella infection and not directly a cause of the shedding event. In the present study of preweaned calves, no associations between the use of antimicrobials or farm- or calf-specific management factors and fecal shedding of multiple-antimicrobial–resistant Salmonella isolates were identified. Because the power of the study was sufficient to detect management factors related to all Salmonella spp, it may be that the entry and presence of multiple-antimicrobial–resistant Salmonella spp are not linked to management practices. Furthermore, most multiple-antimicrobial–resistant Salmonella spp are resistant to antimicrobials commonly used in milk replacer and likely have no influence on the presence of these Salmonella spp in calf feces. AJVR, Vol 67, No. 9, September 2006

Fossler et al10 determined that increased prevalence of Salmonella spp in cows and housing of sick or treated cows in maternity pens at least once per month increased the risk of fecal shedding of Salmonella spp in calves. In the present study, prevalence of Salmonella spp in adult bovids was not assessed and our data could not confirm those findings. Housing of sick animals in maternity pens was uncommon in the present study; only 1 organic dairy housed sick cows with immediate prepartum cows. The finding that the prevalence of fecal shedding of Salmonella spp decreased with calf age may be attributable to age-related changes in commensal enteric flora, a result of which is microflora that is more effective at excluding Salmonella spp than the neonatal microflora. Diet changes during this period could be a reason for the enteric flora shift. Most liquid diets used on dairy farms and calf ranches are energy restricted and alone do not support calf growth during the preweaning period.23 Caloric and protein needs for growth are met by feeding calf starter feeds (primarily grain mixes). As the calf ages and its energy requirements increase, proportionately more of the diet is comprised of starter grain. This may result in changes in the commensal enteric flora. Results of 1 study9 corroborate this finding; an association exists between the early inclusion of hay in calf feed and a lower prevalence of fecal shedding of Salmonella spp.9 Other studies24,25 have revealed that as the calf’s immune system develops, there is reduced fecal shedding of Salmonella spp. Overall, our data suggest that the most important management factor associated with decreased fecal shedding of Salmonella spp by calves was maintenance of a herd that was closed to the introduction of new animals. The inclusion of antimicrobials in the milk replacer was associated with reduced prevalence of fecal shedding of Salmonella spp. Salmonella shedding prevalence was lower as calves aged. Calving or colostrum management did not appear to influence fecal shedding of Salmonella spp in neonatal calves. Antimicrobial use and management and feeding risk factors were not associated with 1587

05-10-0429r.qxp

8/14/2006

3:46 PM

Page 1588

fecal shedding of multiple-antimicrobial–resistant Salmonella spp in preweaned calves. a. b. c. d. e. f. g. h. i.

Copan swab, Copan Diagnostics Inc, Corona, Calif. Difco, Becton Dickinson & Co, Sparks, Md. Hardy Diagnostics, Santa Maria, Calif. Difco Laboratories, Detroit, Mich. ATCC, Manassas, Va. Proc Univariate, version 8.2, SAS Institute Inc, Cary, NC. Proc Cluster, version 8.2, SAS Institute Inc, Cary, NC. Proc Logistic, version 8.2, SAS Institute Inc, Cary, NC. Proc Genmod, version 8.2, SAS Institute Inc, Cary, NC.

References 1. Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999;5:607–625. 2. Berge ACB, Lindeque P, Moore DA, et al. A clinical trial evaluating prophylactic and therapeutic antimicrobial use on health and performance of calves. J Dairy Sci 2004;88:2166–2177. 3. Bulgin MS, Anderson BC, Ward AC, et al. Infectious agents associated with neonatal calf disease in southwestern Idaho and eastern Oregon. J Am Vet Med Assoc 1982;180:1222–1226. 4. Pacer RE, Spika JS, Thurmond MC, et al. Prevalence of Salmonella and multiple antimicrobial-resistant Salmonella in California dairies. J Am Vet Med Assoc 1989;195:59–63. 5. USDA Agricultural Research Service Web site. Bacterial epidemiology and antimicrobial resistance: NARMS reports. Available at: ars.usda.gov/Main/docs.htm?docid=6750. Accessed Jan 1, 2006. 6. Blau DM, McCluskey BJ, Ladely SR, et al. Salmonella in dairy operations in the United States: prevalence and antimicrobial drug susceptibility. J Food Prot 2005;68:696–702. 7. Peek SE, Hartmann FA, Thomas CB, et al. Isolation of Salmonella spp from the environment of dairies without any history of clinical salmonellosis. J Am Vet Med Assoc 2004;225:574–577. 8. Huston CL, Wittum TE, Love BC, et al. Prevalence of fecal shedding of Salmonella spp in dairy herds. J Am Vet Med Assoc 2002;220:645–649. 9. Losinger WC, Wells SJ, Garber LP, et al. Management factors related to Salmonella shedding by dairy heifers. J Dairy Sci 1995;78:2464–2472. 10. Fossler CP, Wells SJ, Kaneene JB, et al. Herd-level factors associated with isolation of Salmonella in a multi-state study of conventional and organic dairy farms II. Salmonella shedding in calves. Prev Vet Med 2005;70:279–291.

1588

11. Hinton M, Suleyman IO, Allen V, et al. Further observations on the excretion of Salmonella in the faeces of calves fed milk substitute. J Hyg (Lond) 1984;93:539–546. 12. Hinton M, Ali EA, Allen V, et al. The excretion of Salmonella typhimurium in the faeces of calves fed milk substitute. J Hyg (Lond) 1983;91:33–45. 13. Bauer AW, Kirby WM, Sherris JC, et al. Antimicrobial susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493–496. 14. Berge ACB, Atwill ER, Sischo WM. Assessing antimicrobial resistance in fecal Escherichia coli in young calves using cluster analysis techniques. Prev Vet Med 2003;61:91–102. 15. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 2nd ed. NCCLS document M31-A2. Wayne, Pa: National Committee for Clinical Laboratory Standards, 2002. 16. Everitt BS, Rabe-Hesketh S. Measures of similarity, dissimilarity and distance. In: The analysis of proximity data. New York: John Wiley & Sons, 1997;11–20. 17. Ward JH. Hierarchical grouping to optimize an objective function. J Am Stat Assoc 1963;58:236–244. 18. Berge ACB, Atwill ER, Sischo WM. Animal and farm influences on the dynamics of antimicrobial resistance in faecal Escherichia coli in young dairy calves. Prev Vet Med 2005;69:25–38. 19. Diez Roux AV. A glossary for multilevel analysis. J Epidemiol Community Health 2002;56:588–594. 20. Agresti A. Analyzing repeated categorical response data. In: Barnett V, Ralph A, Bradley J, et al, eds. Categorical data analysis. 2nd ed. New York: John Wiley & Sons, 2002;386–417. 21. Hosmer DW, Lemeshow S. Logistic regression models for correlated data. In: Hosmer DW, Lemeshow S, eds. Applied logistic regression. 2nd ed. New York: John Wiley & Sons, 2000:308− 330. 22. Lu L, Walker WA. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am J Clin Nutr 2001;73:1124S–1130S. 23. Diaz MC, Van Amburgh ME, Smith JM, et al. Composition of growth of Holstein calves fed milk replacer from birth to 105-kilogram body weight. J Dairy Sci 2001;84:830–842. 24. Mastroeni P, Chabalgoity JA, Dunstan SJ, et al. Salmonella: immune responses and vaccines. Vet J 2001;161:132–164. 25. Mastroeni P. Immunity to systemic Salmonella infections. Curr Mol Med 2002;2:393–406.

AJVR, Vol 67, No. 9, September 2006