Relationship of in vitro minimum inhibitory concentrations of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with signs of bovine respiratory disease

David G. McClary Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Guy H. Loneragan Department of Agricultural Sciences, College of Agriculture, Engineering and Science, West Texas A&M University, Canyon, TX 79016.

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Thomas R. Shryock Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Brandon L. Carter Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Carl A. Guthrie Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Marilyn J. Corbin Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Gerald D. Mechor Elanco Animal Health, 2001 W Main St, Greenfield, IN 46140.

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Abstract

Objective—To determine associations between in vitro minimum inhibitory concentrations (MICs) of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with clinical signs of bovine respiratory disease (BRD).

Design—Observational, retrospective, cohort study.

Animals—976 feeder calves with clinical signs of BRD enrolled in 16 randomized clinical trials.

Procedures—Records of clinical trials from October 26, 1996, to November 15, 2004, were searched to identify calves with BRD from which a single isolate of M haemolytica or P multocida was identified via culture of deep nasal swab samples prior to treatment with tilmicosin (10 mg/kg [4.5 mg/lb], SC) and for which MICs of tilmicosin against the isolate were determined. The MICs of tilmicosin against recovered isolates and response to tilmicosin treatment were evaluated.

Results—Tilmicosin resistance among M haemolytica and P multocida isolates was uncommon (6/745 [0.8%] and 16/231 [6.9%], respectively). Treatment outcome, defined as success or failure after tilmicosin treatment, did not vary with the MIC of tilmicosin against recovered isolates. The proportion of treatment failures attributed to M haemolytica isolates categorized as resistant (MIC of tilmicosin, ≥ 32 μg/mL) or not susceptible (MIC of tilmicosin, ≥ 16 μg/mL), was 0.2% and 0.5%, respectively.

Conclusions and Clinical Relevance—Recovery of tilmicosin-resistant M haemolytica or P multocida isolates was rare, and no association was detected between MIC of tilmicosin and treatment response.

Abstract

Objective—To determine associations between in vitro minimum inhibitory concentrations (MICs) of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with clinical signs of bovine respiratory disease (BRD).

Design—Observational, retrospective, cohort study.

Animals—976 feeder calves with clinical signs of BRD enrolled in 16 randomized clinical trials.

Procedures—Records of clinical trials from October 26, 1996, to November 15, 2004, were searched to identify calves with BRD from which a single isolate of M haemolytica or P multocida was identified via culture of deep nasal swab samples prior to treatment with tilmicosin (10 mg/kg [4.5 mg/lb], SC) and for which MICs of tilmicosin against the isolate were determined. The MICs of tilmicosin against recovered isolates and response to tilmicosin treatment were evaluated.

Results—Tilmicosin resistance among M haemolytica and P multocida isolates was uncommon (6/745 [0.8%] and 16/231 [6.9%], respectively). Treatment outcome, defined as success or failure after tilmicosin treatment, did not vary with the MIC of tilmicosin against recovered isolates. The proportion of treatment failures attributed to M haemolytica isolates categorized as resistant (MIC of tilmicosin, ≥ 32 μg/mL) or not susceptible (MIC of tilmicosin, ≥ 16 μg/mL), was 0.2% and 0.5%, respectively.

Conclusions and Clinical Relevance—Recovery of tilmicosin-resistant M haemolytica or P multocida isolates was rare, and no association was detected between MIC of tilmicosin and treatment response.

Bovine respiratory disease is the most common disease of beef cattle and the most costly disease in terms of economic loss to beef cattle production.1 The most frequently recovered pathogen associated with BRD in feeder cattle is Mannheimia haemolytica, followed by Pasteurella multocida.2 In a study of calves with clinical signs of BRD reported by DeRosa et al,3 investigators demonstrated strong agreement (96%) between isolates of M haemolytica or P multocida obtained from nasal swabs and those obtained from transtracheal tissues.

Despite its causative relationship with BRD, M haemolytica typically inhabits the upper respiratory tract and tonsils in healthy calves and is considered part of the normal upper respiratory tract flora.4–6 It is widely accepted that under conditions of stress or concurrent virus-induced disease, M haemolytica may undergo rapid multiplication and extension beyond the upper respiratory tract into the lower respiratory tract, resulting in bacterial bronchopneumonia.7,8 Likewise, P multocida is considered an important respiratory tract pathogen, although the associated clinical disease and necrotizing pulmonary lesions may not be as severe as those caused by M haemolytica.9

Administration of an effective, approved antimicrobial agent is the most effective method for treatment of BRD.10 Historically, antimicrobial susceptibility of bacterial isolates collected from lungs of calves that died of BRD and submitted to veterinary diagnostic laboratories for bacteriologic culture and susceptibility testing has been used to guide practitioners in selection of antimicrobials in clinical situations.

Standard procedures for conducting antimicrobial susceptibility tests have been described11 by the CLSI. Approved methods for susceptibility testing include agar disk diffusion and microbroth dilution techniques. The Veterinary Antimicrobial Susceptibility Testing subcommittee of the CLSI establishes MIC and disk diffusion clinical breakpoints, also known as interpretive criteria for veterinary antimicrobials.

Antimicrobial clinical breakpoints are established by the CLSI on the basis of 3 components: susceptibility testing of relevant isolates and analysis of scattergrams of the data; the relationship of pretreatment MIC to treatment outcome, usually in clinical efficacy studies; and pharmacokinetic and pharmacodynamic data for the specified antimicrobial drug.12 The purpose of the clinical breakpoint is to assist veterinarians in making informed decisions regarding the selection of antimicrobials for treatment of a specific disease. Categorization of an isolate as susceptible implies that on average an infection may be successfully treated with the appropriate dosage of the antimicrobial agent recommended for that type of infection and infectious species.13 However, resistant isolates are not inhibited or killed by normally achievable concentrations of the antimicrobial under the approved dose regimen or they are more likely to have specific microbial resistance mechanisms; thus, successful treatment is less likely.13

Tilmicosin,a a macrolide antimicrobial drug, was approved in the United States for treatment of BRD caused by M haemolytica in 1992. Numerous studies14–18 have confirmed the benefits of tilmicosin treatment in calves with BRD. The CLSI-established MIC breakpoints18 of tilmicosin for M haemolytica are 8, 16, and 32 μg/mL for susceptible, intermediate, and resistant isolates, respectively; these were established by use of the same methods used in the study reported here. Although there are no CLSI-established breakpoints of tilmicosin for P multocida, 8, 16, and 32 μg/mL would be considered appropriate breakpoints for that microorganism as well.19

In addition to bacterial resistance mechanisms, various causes of treatment failures, such as death of treated cattle and relapses of BRD, have been reported.10 Reasons for treatment failure include administration of treatment late in the course of the disease, incorrect diagnosis, and other underlying disease conditions contributing to a compromised immune system. Few reports are available that compare the established MIC of an antimicrobial drug for a specific microorganism with outcomes in food-producing animals following treatment with that drug for infection. Consequently, the association between in vitro bacterial susceptibility to some antimicrobial drugs and clinical outcome has been questioned because treatment of cattle infected with apparently susceptible isolates was associated with poor clinical outcomes or treatment of cattle infected with isolates determined to be resistant to a specific antimicrobial was associated with favorable outcomes.20,21 The purpose of the study reported here was to test the null hypothesis that there would be no association between MICs of tilmicosin in vitro against isolates of M haemolytica, P multocida, and Histophilus somni (collected via DNS from feeder calves with clinical signs of BRD prior to treatment) and tilmicosin treatment outcome.

Materials and Methods

Study population and record assessment—Records of 16 randomized clinical trials that were conducted in confined cattle feeding facilities in 7 states from October 26, 1996, to November 15, 2004, were searched to identify feeder calves that received tilmicosin for treatment of BRD (Table 1). Records of various types and breeds of beef feeder calves (6 to 10 months old) that had no history of previous antimicrobial treatment, including metaphylaxis, and met criteria for the study definition of BRD (clinical signs of BRD [CIS, 2 to 5 on a 1 to 5 scale as described elsewhere22] with a rectal temperature ≥ 40.0°C [104.0°F]) at the time of enrollment were included in the present study. In all trials, DNS samples were collected from all eligible cattle prior to antimicrobial treatment and susceptibility status of the isolates was not known or considered in the treatment decision. In each of the trials, the day of treatment was considered day 0; if the CIS of a calf was not lower on day 3 after treatment, compared with the CIS at enrollment, and rectal temperature was again ≥ 40.0°C, the case was identified as a treatment failure. If the CIS on day 3 was improved but the calf met criteria for BRD again ≤ 21 days after treatment, the case was considered a relapse of BRD. If the calf had no clinical signs of BRD ≤ 21 days after treatment and did not die or require euthanasia because of BRD prior to conclusion of the clinical trial, the case was considered a treatment success. The clinical trials were funded by a drug companya and were conducted according to the various contractors' established animal care protocols. For analytic purposes in the present study, cases identified as treatment failures and BRD relapses were combined to represent an unsuccessful treatment outcome.

Table 1

Locations and years of randomized clinical trials with numbers of calves that had Mannheimia haemolytica and Pasteurella multocida identified as single isolates in DNS samples from 976 feeder calves* with clinical signs of BRD.

   Single isolate 
Trial No.StateYearM hemolytica (No. of calves)P multocida (No. of calves)
1Idaho19961713
2Texas1997582
3Idaho1998773
4Nebraska1998483
5Idaho19995821
6Idaho19991621
7Idaho20006126
8Texas20007732
9Kansas20011210
10Mississippi20014320
11Arkansas2001910
12Texas20012619
13Colorado2002176
14Nebraska2002727
15Texas20034513
16Colorado200410925
Total  745231

Records of randomized clinical trials were reviewed in a retrospective cohort study. All DNS samples were obtained prior to treatment of calves with tilmicosin (10 mg/kg [4.5 mg/lb], SC).

Only cases in which a single isolate of the organism was identified in culture were included in the study; those in which multiple phenotypically distinct isolates of 1 organism or single isolates of > 1 organism were identified were excluded.

Sample collection and treatment—All calves in each of the trials that met the case definition for BRD were restrained in a cattle chute for DNS sample collection immediately prior to antimicrobial administration (for purposes of the study reported here, the treatment selected was tilmicosin,b (10 mg/kg [4.5 mg/lb], SC). A DNS sample was collected by use of an 80-cm long, cotton-tipped, guarded swabc passed via one of the nares through the ventral nasal meatus to the pharyngeal area. After withdrawal into the self-contained transport tip, the tip was snapped off according to the manufacturer's instructions and plugged on each end with plastic plugs provided with the swab. Each sample was labeled and stored on ice or refrigerated at 4°C until transported by courier service (within 24 hours after collection) to microbiology laboratoriesd–k near each of the individual clinical trial sites.

Microbiological analysis—At each microbiology laboratory,d–k samples were placed on 5% sheep blood or 5% bovine blood agar plates. Samples were incubated for ≥ 18 hours at 36 ± 2°C in 5 ± 2% CO2. Plates were examined for the presence of respiratory pathogens, including colonies of M haemolytica, P multocida, and H somni, by use of standard identification procedures. The identity of each isolate of interest was confirmed by means of Gram staining, oxidase and indole biochemical testing, atmospheric CO enrichment, and growth on MacConkey agar as appropriate. Quality-control strains of representative bacteria were included with each identification test performed.

Following pathogen isolation and identification, samples with positive test results were transported by courier from the laboratory in which the microorganisms were first isolated to one of 2 laboratories,d,f where MICs of tilmicosin against each isolate were determined. Both laboratories followed CLSI procedures11 for MIC testing in cation-adjusted Mueller-Hinton broth for M haemolytica and P multocida isolates and in veterinary fastidious medium for H somni. Personnel at one microbiology laboratoryd used commercially available microdilution plates,l and those at the otherf used plates prepared on the premises that contained 100 μL of cation-adjusted Mueller-Hinton broth or veterinary fastidious medium. Inoculated plates that contained various concentrations of tilmicosin (range, 0.5 to 64.0 μg/mL) were incubated aerobically at 36 ± 2°C for 16 to 20 hours, except for H somni–inoculated plates, which were incubated in a 5 ± 2% CO2 atmosphere at 36 ± 2°C for 16 to 20 hours. Microorganisms included for quality-control purposes included Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 29213); these were tested on each sample testing date. To assure proper quality control when the MIC of tilmicosin against H somni isolates was determined, H somni (ATCC 700025) was tested concurrently. Positive growth controls, negative sterility controls, and inoculum counts were also included.

Statistical analysis—Data were combined into a single electronic data setm and validated. Data were then imported into a commercially available software package for analysis.n Only data for calves treated with tilmicosin after DNS sample collection from which a single, well-defined isolate of M haemolytica or P multocida was recovered were included in the statistical analysis. When ≥ 2 phenotypically distinct isolates of M haemolytica or ≥ 2 phenotypically distinct isolates of P multocida were recovered from a sample, or 1 isolate each of M haemolytica and P multocida was recovered from a sample, data for that calf were excluded from the analysis. Because a relatively small number of samples met the inclusion criteria, isolates for which the MIC of tilmicosin was 0.5, 1.0, or 2 μg/mL were combined into a single category (MIC ≤ 2 μg/mL). The MICs of tilmicosin against all isolates were then log2 transformed. Initial descriptive statistics, such as distribution of M haemolytica and P multocida among calves from the various clinical trials, were generated and inspected prior to model development. Various generalized linear mixed models were constructed to evaluate the association between treatment success (binary response variable) and susceptibility of the recovered organism to tilmicosin; in separate models, log MIC was treated as either a classification variable or continuous variable. In further models, recovery of an isolate categorized as not susceptible (ie, intermediate or resistant [MIC of tilmicosin, ≥ 16 μg/mL; yes vs no]) and recovery of a resistant isolate (MIC of tilmicosin, ≥ 32 μg/mL; yes vs no) were modeled as independent variables. A random effect of clinical trial was included in the model because outcome may have been clustered within a trial. The choice of an unstructured within-trial covariance matrix was made on the basis of model fit criteria (ie, the nested model with the lowest Akaike information criterion was selected). Intercept-only models were constructed to calculate overall probability of treatment success after accounting for within-trial clustering. A model with outcome as the dependent variable and pathogen as the independent variable was constructed. Where appropriate (treating MIC of tilmicosin as a class variable with a significant P value), odds of treatment success were compared. Odds of treatment success and 95% CIs were back-transformed to a proportion and graphed.o Models were constructed according to the pathogen that was recovered (M haemolytica or P multocida). Histophilus somni—positive samples were excluded from the analysis because of sparse data.

Population attributable fraction,23 an estimate of the proportion of total treatment failures attributable to antimicrobial susceptibility of the organism, was calculated. Model-adjusted relative risks (from models of M haemolytica and P multocida) were calculated from model-adjusted, back-transformed risk of treatment failure, and PAF was calculated by use of the following equation:

article image

where p is the proportion of all calves from which a resistant isolate was recovered or, in separate calculations, the proportion of calves in which the isolate was categorized as not susceptible (ie, intermediate or resistant).

Results

A total of 1,237 M haemolytica (n = 878) and P multocida (359) isolates were collected from 1,100 calves with clinical signs of BRD during 16 clinical trials. Histophilus somni was isolated from only 29 (2.6%) calves that had clinical signs of BRD; this was the only isolate recovered in 17 cases, whereas in the other 12, H somni was isolated in combination with either M haemolytica or P multocida. Some interyear and intertrial variation was observed for relative numbers of M haemolytica to P multocida isolates. Overall, 71.0% of the isolates were identified as M haemolytica; the lowest M haemolytica: P multocida ratio detected was 43.2:56.8, and the highest ratio was 96.7:3.3. Of the 1,100 calves, samples from 976 were included in the analysis (ie, yielded a single, well-defined bacterial isolate). Of these 976 samples, 745 yielded a single M haemolytica isolate (Table 2) and 231 yielded a single P multocida isolate (Table 3).

Table 2

Number (percentage) of single isolates of M haemolytica that were recovered from DNS samples of 745 feeder calves* with clinical signs of BRD and categorized according to MIC of tilmicosin in vitro.

  MIC (μg/mL)       
TrialYear≤ 0.5124816≥ 32Total
119960(0)0(0)0(0)15(88.2)2(11.8)0(0)0(0)17
219970(0)0(0)19(32.8)36(62.1)3(5.2)0(0)0(0)58
319980(0)1(1.3)68(88.3)8(10.4)0(0)0(0)0(0)77
419980(0)0(0)47(97.9)0(0)1(2.1)0(0)0(0)48
519990(0)0(0)8(13.8)46(79.3)3(5.2)0(0)1(1.7)58
619990(0)1(6.3)5(31.3)8(50.0)2(12.5)0(0)0(0)16
720000(0)5(8.2)41(67.2)9(14.8)4(6.6)1(1.6)1(1.6)61
820000(0)2(2.6)19(24.7)51(66.2)4(5.2)0(0)1(1.3)77
920010(0)0(0)9(75.0)2(16.7)0(0)0(0)1(8.3)12
1020010(0)1(2.3)8(18.6)15(34.9)14(32.6)4(9.3)1(2.3)43
1120010(0)0(0)0(0)5(55.6)2(22.2)2(22.2)0(0)9
1220010(0)1(3.8)15(57.7)8(30.8)2(7.7)0(0)0(0)26
1320020(0)0(0)1(5.9)16(94.1)0(0)0(0)0(0)17
1420020(0)0(0)2(2.8)37(51.4)10(13.9)22(30.6)1(1.4)72
1520030(0)0(0)2(4.4)20(44.4)4(8.9)19(42.2)0(0)45
1620040(0)0(0)39(35.8)66(60.6)1(0.9)3(2.8)0(0)109
Total 0(0)11(1.5)283(38.0)342(45.9)52(7.0)51(6.8)6(0.8)745

Percentages are relative to the total reported for each row.

See Table 1 for remainder of key.

Table 3

Number (percentage) of single isolates of P multocida that were recovered from DNS samples of 231 calves* with clinical signs of BRD and categorized according to MIC of tilmicosin.

  MIC (μg/mL)       
TrialYear≤ 0.5124816≥ 32Total
119961(7.7)0(0)2(15.4)2(15.4)6(46.2)1(7.7)1(7.7)13
219970(0)0(0)1(50.0)1(50.0)0(0)0(0)0(0)2
319981(33.3)1(33.3)1(33.3)0(0)0(0)0(0)0(0)3
419980(0)0(0)0(0)0(0)0(0)2(66.7)1(33.3)3
519990(0)2(9.5)18(85.7)1(4.8)0(0)0(0)0(0)21
619991(4.8)1(0)16(85.7)3(9.5)0(0)0(0)0(0)21
720001(3.8)7(26.9)7(26.9)7(26.9)4(15.4)0(0)0(0)26
820000(0)3(9.4)11(34.4)9(28.1)3(9.4)3(9.4)3(9.4)32
920010(0)0(0)0(0)5(50.0)2(20.0)0(0)3(30.0)10
1020010(0)0(0)0(0)14(70.0)4(20.0)2(10.0)0(0)20
1120010(0)0(0)0(0)4(40.0)4(40.0)0(0)2(20.0)10
1220010(0)0(0)8(42.1)5(26.3)5(26.3)0(0)1(5.3)19
1320020(0)0(0)2(33.3)3(50.0)1(16.7)0(0)0(0)6
1420020(0)0(0)0(0)1(14.3)1(14.3)0(0)5(71.4)7
1520031(7.7)0(0)2(15.4)3(23.1)3(23.1)4(30.8)0(0)13
1620041(4.0)2(8.0)10(40.0)9(36.0)1(4.0)2(8.0)0(0)25
Total 6(2.6)16(6.9)78(33.8)67(28.6)34(14.7)14(6.1)16(6.9)231

See Tables 1 and 2 for key.

Minimum inhibitory concentration distributions were variable among trials. The overall MIC of tilmicosin required to inhibit growth of 50% of M haemolytica and P multocida organisms was 4 μg/mL, whereas the MIC required to inhibit growth of 90% of organisms was 8 μg/mL for M haemolytica and 16 μg/mL for P multocida. On the basis of results of tilmicosin testing, recovered M haemolytica and P multocida organisms were categorized as susceptible, not susceptible, or resistant (MICs, ≤ 8 μg/mL, ≥ 16 μg/mL, or ≥ 32 μg/mL, respectively). Only 6 of 745 (0.8%) M haemolytica and 16 of 231 (6.9%) P multocida isolates were phenotypically resistant to tilmicosin (Tables 2 and 3). Most (42/57 [73.7%]) of the M haemolytica isolates categorized as either not susceptible or resistant were obtained during 2 trials (1 trial in Nebraska in 2002 and 1 in Texas in 2003). Reference strains were all within acceptable quality-control ranges for each testing period.

Overall probability of successful treatment was 58.1% for calves that had a single M haemolytica isolate and 60.2% for calves that had a single P multocida isolate. After adjustment for within-trial clustering, model-adjusted estimates of the probability of treatment success were 61.0% and 63.6% for those calves from which either an M haemolytica or P multocida isolate was recovered, respectively. However, there was no significant difference between likelihood of treatment success and pathogen type recovered (P = 0.27).

For individual calves from which an M haemolytica isolate was recovered, no association was detected between MIC of tilmicosin and treatment response either on a linear scale (OR, 1.08; 95% CI, 0.87 to 1.34; P = 0.47) or as a classification variable (P = 0.45; Figure 1). Furthermore, we did not detect an association between treatment outcome and recovery of a susceptible isolate, compared with recovery of an isolate that was not susceptible (OR, 0.55; 95% CI, 0.27 to 1.08; P = 0.08; Figure 2). Additionally, no association was detected (OR, 0.40; 95% CI, 0.06 to 2.59; P = 0.34) between treatment outcome and recovery of a resistant isolate, compared with recovery of an isolate that was not resistant (ie, < 32 μg/mL). The proportion of treatment failures attributable to recovery of an M haemolytica isolate that was categorized as resistant or not susceptible (ie, population attributable fraction) was 0.2% and 0.5%, respectively.

Figure 1—
Figure 1—

Probability of treatment success determined in a retrospective cohort study of 976 feeder calves with clinical signs of BRD from which a single isolate of Mannheimia haemolytica (black bars) or Pasteurella multocida (white bars) was recovered from DNS samples. Isolates were categorized according to MIC of tilmicosin prior to administration of the drug (10 mg/kg [4.5 mg/lb], SC). Error bars represent upper 95% CI. Numbers above the error bars represent number of isolates within each category.

Citation: Journal of the American Veterinary Medical Association 239, 1; 10.2460/javma.239.1.129

Figure 2—
Figure 2—

Probability of treatment success determined in the 976 calves in Figure 1 with isolates of M haemolytica (black bars) or P multocida (white bars) categorized on the basis of results of tilmicosin testing as susceptible (MIC ≤ 8 μg/mL), not susceptible (MIC ≥ 16 μg/mL), or resistant (MIC ≥ 32 μg/mL). See Figure 1 for remainder of key.

Citation: Journal of the American Veterinary Medical Association 239, 1; 10.2460/javma.239.1.129

For calves from which a single P multocida isolate was recovered, no association was detected between MIC of tilmicosin and treatment outcome either on a linear scale (OR, 0.86; 95% CI, 0.61 to 1.19; P = 0.36) or as a classification variable (P = 0.64; Figure 1). Furthermore, as observed in the analysis of M haemolytica isolates, we did not detect an association between recovery of an isolate that was not susceptible (OR, 1.51; 95% CI, 0.54 to 4.25; P = 0.43) or was resistant (OR, 1.08; 95% CI, 0.28 to 4.13; P = 0.91) with treatment outcome (Figure 2).

Discussion

Results of the study reported here indicated M haemolytica was the most commonly (745/976 [76.3%]) isolated microorganism from calves with clinical signs of BRD in cases for which a single isolate was recovered. Furthermore, the distribution ratio of M haemolytica to P multocida did not appear to vary substantially among years or among various breeds and types of beef feeder cattle included in the trial populations of calves. This observation differs somewhat from the finding of Welsh et al24 that the ratio of P multocida to M haemolytica increased between 1998 and 2002. In the 16 clinical trials from which data for the present study were obtained, the number of clinical infections associated with a single isolate was greater for M haemolytica (745/878 [84.9%]) than for P multocida (231/359 [64.3%]), possibly indicating P multocida was more likely to be opportunistic or a copathogen rather than a primary pathogen. This observation is consistent with those of other studies9 in that P multocida produced less severe pulmonary lesions, compared with M haemolytica, and that P multocida could be isolated from pneumonic lungs in which it was not the primary pathogen.

In the clinical trials that provided records for use in the present study, all calves were treated with tilmicosin without prior knowledge of the susceptibility status of the bacterial isolates. The distribution of MICs of tilmicosin did not appear to differ between M haemolytica and P multocida isolates. Furthermore, the prevalence of tilmicosin-resistant M haemolytica (6/745 [0.8%]) and P multocida (16/231 [6.9%]) isolates from pretreatment DNS specimens was low, indicating the use of macrolides in cattle over several decades has not generated a reservoir of macrolide-resistant BRD-associated pathogens detectable by use of nonselective surveillance methods.

Tilmicosin breakpoints for M haemolytica have been applied since 1996. In conjunction with clinical experience, these are intended to be used as a guide in selection of appropriate antimicrobial treatment for microbial infections. Typical expectations in the field are that calves from which isolates with lower MICs are recovered are more likely to have a favorable clinical response to treatment, compared with calves from which an isolate with a higher MIC is recovered. However, in the analyses of the data reported here, MICs of tilmicosin were of limited predictive value in terms of clinical outcome of calves from various populations of cattle treated with this drug. This observation is similar to those reported in other studies in which antimicrobial susceptibility testing was of limited value in terms of predicting therapeutic outcome to treatment of mastitis with oxytetracycline20 or pirlimycin hydrochloride.21

Although there appeared to be some evidence of greater treatment success against M haemolytica isolates classified as susceptible to tilmicosin, compared with those determined to be not susceptible (ie, categorized as either intermediate or resistant), this difference was not statistically significant (P = 0.08). The comparison of treatment outcome and recovery of a resistant versus not resistant (ie, categorized as either intermediate or susceptible) isolate was associated with a much larger P value (ie, P = 0.34); however, this comparison lacked statistical power as there were only 6 isolates that fell into the resistant category. Wide CIs (Figures 1 and 2) indicate this lack of precision and statistical power. Although the observed relationship is biologically plausible, the evidence supporting it is weak and may be subject to type I error; a potential association between MIC and treatment outcome warrants further studies that have greater statistical power to more appropriately test this hypothesis regarding treatment outcomes for M haemolytica–associated BRD. On the other hand, there was clearly no evidence for an association between the MIC of tilmicosin against P multocida and treatment outcome in models that had substantially greater power.

Clinical breakpoints of an antimicrobial against a particular bacterial pathogen are one piece of information for the clinician to consider when selecting an antimicrobial for treatment of a disease condition. However, it is important to recognize that clinical breakpoints are not intended to be predictive of the clinical outcome for an individual calf with BRD because of the multiplicity of host-pathogen-antimicrobial interactions.

Possible explanations for the failure to detect an association between the MIC of tilmicosin against isolated organisms and treatment outcome for calves reported in these trials were that isolates recovered in pure culture from a DNS were not good indicators of the microbial population within lung tissue of calves with BRD or that calves with clinical signs of BRD may have had a mixed microbial population in the lungs. Thus, classification of the population as resistant or susceptible on the basis of results of a single sample culture may result in erroneous conclusions. If this misclassification is nondifferential, then the data were by definition biased toward the null hypothesis. Another possibility is that use of the CIS method resulted in some amount of non-differential misclassification (ie, calves that no longer had clinical signs of BRD classified as treatment failures or calves that had clinical signs classified as treatment successes), which would also bias the data toward the null hypothesis, and as previously discussed, lack of statistical power may have been an important consideration. Although at least 1 study3 of calves with clinical signs of respiratory disease revealed an association between recovery of respiratory tract pathogens from nasal swabs and recovery of the same pathogens from transtracheal swabs of the same animal, additional investigations should be performed in this area.

The population attributable fraction, a measure of the proportion of all observed treatment failures attributable to classification of an M haemolytica isolate recovered from a DNS as either not susceptible or resistant on the basis of results of tilmicosin testing, was 0.5% and 0.2% for the 2 categories, respectively. In other words, the vast majority of treatment failures were likely attributable to factors other than macrolide resistance among isolates recovered from the DNS. Although these estimates were likely biased somewhat toward 0% because of nondifferential misclassification, a better understanding of other potential variables associated with treatment failure, especially those related to calves' intrinsic mechanisms to resist or overcome BRD, could help to improve predictions of treatment outcome.

One important factor is the ability of the calf to effectively respond immunologically to growing microbial populations within the lower respiratory tract. If a calf was immunocompromised because of factors such as stress, poor nutritional status, concurrent infection with a viral pathogen, or lack of prior exposure to the pathogen, the clinical response would likely have been poor regardless of the susceptibility of the pathogen to the antimicrobial used. Alternatively, if the immune system was competent and the calf minimally stressed, the clinical response may have been favorable even if the pathogen was resistant to the antimicrobial used. Other factors (not evaluated in the present study) that may have a role in determining treatment success or failure include the timing of treatment initiation relative to development of BRD. If treatment was initiated after the development of severe lung disease, results were likely to be poor regardless of the susceptibility of the associated pathogen. It may also have been possible that other unmeasured factors resulted in differential misclassification or confounding and biased the measures of effect in an uncertain direction in relation to the null hypothesis.

ABBREVIATIONS

ATCC

AmericanType Culture Collection

BRD

Bovine respiratory disease

CI

Confidence interval

CIS

Clinical illness score

CLSI

Clinical and Laboratory Standards Institute

DNS

Deep nasal swab

MIC

Minimum inhibitory concentration

OR

Odds ratio

a.

Elanco Animal Health, Greenfield, Ind.

b.

Micotil 300, Elanco Animal Health, Greenfield, Ind.

c.

Accu-Culshure, Accu-Med Corp, Pleasantville, NY.

d.

Elanco Animal Health Microbiology Laboratory, Greenfield, Ind.

e.

Texas Veterinary Medical Diagnostic Laboratory System, Amarillo, Tex.

f.

Microbial Research Inc, Fort Collins, Colo.

g.

Kansas State University, Veterinary Diagnostic Laboratory, Manhattan, Kan.

h.

Mississippi State Veterinary Diagnostic Laboratory, Jackson, Miss.

i.

Midwest Veterinary Services, Oakland, Neb.

j.

University of Idaho, Caine Veterinary Teaching Center, Caldwell, Idaho.

k.

University of Arkansas Microbiology Laboratory, Fayetteville, Ariz.

l.

Sensititre, Somagen Diagnostics, Edmonton, AB, Canada.

m.

Microsoft Excel, Microsoft Corp, Redmond, Wash.

n.

SAS, version 9.2, SAS Institute Inc, Cary, NC.

o.

SigmaPlot, release 10.0, Systat, San Jose, Calif.

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