Materials and Methods

Selection of beef herds—The study was conducted from May through November 2004. Individual producers were contacted via telephone to solicit participation in the study. They were included in the study if they owned a backgrounding or stocker operation, provided information on when a new shipment of calves would arrive, and agreed to have samples taken while they processed the calves up to 10 days after arrival. A backgrounding operation was defined as an operation that purchased weaned beef calves from various sources and processed them for sale and shipment to western US feedlots. A stocker operation was defined as an operation that purchased weaned beef calves, processed them, and fed them on grass pasture for varying lengths of time before sale and shipment to western feedlots. Nine operations were enrolled in the study. Because several of the operations could be classified as having both backgrounded and stocker cattle, the operations were referred to as backgrounding-stocker operations. Seven of the operations were in northern Georgia, and 2 of the operations were in southern Georgia.

Sample collection and clinical examination—Calves sampled were 3 to 9 months of age. Forty calves from farm 1, 50 calves each from farms 2 through 8, and 42 calves from farm 9 were sampled from 1 to 10 days after arrival. Samples were collected at the time of processing. Processing involved any of the following: administration of antimicrobials, vaccines, vitamin and mineral supplements, or antiparasitic drugs; dehorning; castration; ear tag placement; or tattooing. Calves were from multiple sources (often purchased through sale barns), and it was not possible to confirm the location from which the calves originated prior to purchase by the backgrounding-stocker operation.

Two nasal swab specimens were collected from each calf. Swabs were simultaneously inserted into the nasal passage and then placed into transport media.^a Rectal temperature was measured in each calf, and the presence and consistency of any nasal discharge was recorded. Any obvious abnormality involving the ocular, respiratory, or musculoskeletal systems was also recorded.

Samples were transported on ice to the laboratory for processing. One swab specimen was used for DNA extraction and PCR assay, and the other was used for bacteriologic culture. The DNA extraction swab specimens were processed immediately or kept on ice (< 4°C) for up to 48 hours. The swab specimens for bacteriologic culture were processed immediately or kept on ice (< 4°C) for up to 36 hours.

Bacteriologic culture—Modified Hayflick media supplemented with horse serum, yeast extract, glucose, penicillin, and thallium acetate was used for culture of swab specimens. Swabs were treated in 2 ways during the course of the study. Swabs were removed from the transport media and placed directly into broth for farms 1 through 4 or streaked onto plates and then placed into broth for farms 5 through 9. For farms 1 and 2, swabs were placed in broth, then 1 mL of broth was filtered by use of a 0.45-Mm filter into a tube of fresh broth. Broth cultures were incubated at 37°C with 5% CO₂ and monitored daily for a pH change caused by bacterial growth, as evidenced by a change in color from red to yellow; when color change was evident, a 10-μL loop of sample was streaked onto plates. If no color change occurred within 10 days, broth samples were plated after 10 days of incubation.

Once placed on plates, the samples were incubated at 37°C with 5% CO₂. The plates were checked every 24 to 48 hours for growth. If no growth was observed in 21 days, results were considered negative and plates were discarded. Results were considered positive for growth of mycoplasmas if pinpoint colonies typical of Mollicutes were grossly visible. Pinpoint colonies were viewed with an inverted light microscope for characteristic colony appearance. Colonies were confirmed to be Mollicutes and identified specifically as M bovis via PCR assay of DNA isolated from individual colonies. Plates were discarded if large bacterial colonies considered non-Mollicutes were seen. Individual colonies were picked off the plates, placed into 100 μL of mycoplasma-free water (autoclaved ultrapure water), and stored at −20°C for DNA extraction. Colonies from the original plate were then reisolated on a second plate to create a homogeneous population. Several quarter-inch cubes of agar were cut out from areas with heavy growth and placed into cryovials and stored at −70°C.

DNA extraction and PCR assay—For direct identification of Mollicutes and M bovis, DNA was extracted from the second swab specimen with a commercially available DNA extraction kit^b by use of the manufacturer's instructions for the buccal swab spin protocol, except that 400 μL of deionized water was replaced with mycoplasma-free water and the centrifugation step following buffer AW2 addition at 20,000 X g for 3 minutes was replaced with 16,000 X g for 5.5 minutes. Deoxyribonucleic acid was also extracted from colonies isolated via culture. For long-term DNA storage, the samples were frozen at −20°C. The samples underwent 2 rounds of PCR, 1 by use of primers specific for the class Mollicutes and the second by use of primers specific for M bovis. A 580-bp DNA segment specific for Mollicutes was amplified by use of primer pair MW28 (5′-CCA GAC TCC TAC GGG AGG CA-3′) and MW29 (5′-TGC GAG CAT ACT ACT CAG GC-3′) as described.¹⁴ A 360-bp DNA segment specific for M bovis was amplified by use of primers MboF (5′-CCT TTT AGA TTG GGA TAG CGG ATG-3′) and MboR (5′-CCG TCA AGG TAG CAT CAT TTC CTA T-3′) as described.¹⁵ Each PCR reaction was carried out with 1 μL of template and 9 μL of master mix containing 0.2 μL of dNTP (10mM), 0.1 μL of DNA polymerase^c (5 U/μL), 6.7 μL of dH20, 1 μL of MgCl (20mM), and 0.5 μL of each forward and reverse primer (50 pmol). The PCR reactions for Mollicutes and M bovis were run at 94°C for 30 seconds, 55°C for 10 seconds, and 72°C for 35 seconds for 30 cycles. For the negative control, DNA was replaced with mycoplasma-free water. Deoxyribonucleic acid samples from Mycoplasma bovigenitalium or Mycoplasma bovirhinis were used as positive controls for Mollicutes PCR assays. Deoxyribonucleic acid from M bovis was used as the positive control for species PCR assays. Amplicons were examined by use of electrophoresis in 1.5% agarose gels, stained with ethidium bromide, electrophoresed for 45 minutes at 110 V, and examined with a UV lamp.

Because there was a chance that primers originally selected to identify M bovis could cross-amplify Mycoplasma agalactiae,¹⁵ a final confirmatory PCR assay was performed on all samples with positive results for M bovis by use of primers MBOUVRC2-L (5′-TTA CGC AAG AGA ATG CTT CA-3′) and MBOUVRC2-R (5′-TAG GAA AGC ACC CTA TTG AT-3′), which yielded a 1,626-bp fragment, as described, and reliably differentiated M bovis from M agalactiae.¹⁶ The amplification cycles were as follows: 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute for 35 cycles. Deoxyribonucleic acid from M bovis was used as the positive control. Amplicons were examined via electrophoresis in 1.5% agarose gels, stained with ethidium bromide, electrophoresed for 45 minutes at 110 V, and examined with a UV lamp.

Statistical analysis—Parallel test interpretation was used to classify calves as having positive results for M bovis if the nasal swab specimen yielded positive results by use of either culture or PCR assay.¹⁷ To determine whether calves shedding M bovis were more likely to have fever (rectal temperature > 39.7°C [103.5°F]) or mucopurulent nasal discharge than were calves not shedding M bovis, the proportion of all calves sampled that had these signs and positive results for M bovis by either culture or PCR assay were compared with calves with negative results for M bovis by use of the Fisher exact test and calculation of prevalence odds ratios. The 95% CI was calculated by use of the approximation of Woolf.^d The kappa statistic was calculated to assess the agreement between results of culture and PCR assay for M bovis for individual- and farm-level determination of M bovis status.¹⁸

Results

Four hundred thirty-two calves were sampled at 9 operations. Of the 432 calves sampled, 374 (87%) yielded positive results for Mollicutes via PCR assay and 63 (15%) via bacteriologic culture. At the farm level, prevalence of Mollicutes ranged from 58% to 100% as measured via PCR assay and from 0% to 59% as measured via culture.^e Seven of the 432 (2%) calves sampled had positive results for M bovis via PCR assay, and 10 (2%) had positive results for M bovis via culture. Prevalence of M bovis at the farm level ranged from 0% to 4% via PCR assay and from 0% to 6% via culture.^e

Of the 9 farms sampled, 6 yielded at least 1 calf with positive results for M bovis. Because there was not a notable difference in prevalence for farms 6 through 9, compared with farms 1 through 5, it appeared that the slight change in culture methodology made for farms 6 through 9, wherein the swab specimen was streaked on a plate before being placed into broth, made no difference in rate of isolation of M bovis. Overall, 12 calves had positive results for M bovis by use of either PCR assay or culture. Seven of 12 had positive results via PCR assay, and 10 of 12 had positive results via culture. Five of 12 calves had positive results via both PCR assay and culture. The kappa statistic for agreement between culture and PCR assay at the calf level was 0.58, indicating moderate agreement for identification of individual calves shedding M bovis. However, when at least 1 calf on a given farm had positive results via culture, there was also at least 1 calf with positive results via PCR assay, indicating complete agreement between culture and PCR assay for finding farms with positive results for M bovis.

The number and percentage of calves with positive results for Mollicutes or M bovis via PCR assay or culture that also had fever or mucopurulent nasal discharge were determined.^e In general, calves were more likely to have fever than mucopurulent nasal discharge if they had positive results for Mollicutes or M bovis. Calves shedding M bovis as measured by either PCR assay or culture had significantly increased odds of fever, compared with calves not shedding M bovis. For calves with positive results for M bovis via PCR assay, the odds ratio for fever was 10.6 (P = 0.01; 95% CI, 1.3 to 88.6); for calves with positive results for M bovis via culture, the odds ratio for fever was 7.1 (P < 0.01; 95% CI, 1.5 to 33.9). The odds of mucopurulent nasal discharge were not significantly greater for calves with positive results for M bovis via either PCR assay or culture, compared with calves with negative results. No other obvious abnormalities were detected in any calf.

Discussion

Mycoplasma bovis has been isolated from dairy and beef calves with respiratory disease in multiple studies,^2-4,12,19,20 with particularly high prevalence in calves with chronic pneumonia nonresponsive to antimicrobial treatment.^1–5 The prevalence of nasal shedding of M bovis in herds of healthy cattle is less well characterized, and to our knowledge, this report is the first description of nasal shedding in calves entering backgrounding or stocker operations in the southeastern United States. Studies^8,9,11 of shedding of M bovis by dairy calves with no signs of respiratory disease have indicated prevalence at the calf level of 0% to 30%. Backgrounding and stocker operations are the source of many cattle that subsequently enter feedlots, and M bovis is an important cause of disease in feedlot cattle.^1,5,21 However, almost nothing is known about how M bovis infection and transmission in backgroundingstocker operations influence disease in feedlots that receive calves from these operations. The present study provides an important basis for future research to determine how circulation of M bovis relates to disease in backgrounding-stocker operations, as well as the relationship between exposure and disease in these operations and disease in feedlots that receive such calves.

In the present study, a large proportion of calves had positive results for organisms of the Mollicutes class via PCR assay and culture of nasal swab specimens. In contrast, relatively few calves had positive results for M bovis, indicating that nasal shedding of M bovis occurred at a low level in recently purchased calves on backgrounding-stocker operations in Georgia. It was not surprising to find that a large proportion of the calves had positive results for Mollicutes because these organisms are common inhabitants of the respiratory tract of cattle and many appear to be nonpathogenic commensals.^7,22

Although M bovis can be found in nasal secretions of clinically normal cattle,^8–11 in this study, fever was significantly more common in calves shedding M bovis than in calves not shedding M bovis. The importance of fever in calves shedding M bovis cannot be definitively established; the association may have been because calves shedding M bovis had an infectious or inflammatory condition that enhanced colonization with M bovis, or it may have indicated that calves were in some stage of disease attributable to M bovis. Other pathogens were not identified in these calves, so it is also possible that the fever could have been caused by other coinfecting organisms.

Results of the present study indicated that PCR assay can be a fast and accurate test for identifying whether cattle on a given farm are shedding M bovis. Notably, there was excellent agreement between culture and PCR assay at the farm level; on 3 farms, no calves had positive results for M bovis shedding via culture or PCR assay, and whenever at least 1 calf had positive results via culture for M bovis on a given farm, at least 1 calf also had positive results via PCR assay. However, agreement between results of PCR assay and culture was only moderate at the individual calf level, and slightly more calves were identified as M bovis shedders via culture than via PCR assay. Imperfect agreement between culture and PCR assay at the calf level could have been influenced by any of the following factors: the use of a dual swab may have resulted in 1 swab collecting bacteria or bacterial DNA, whereas the other swab did not; heavy bacterial load or debris on the swab may have interfered with PCR assay; and small numbers of bacteria or bacterial DNA may have precluded identification of organisms and DNA. These factors may have been important individually or in an additive manner.

It is important to note that the herds sampled in this study were not having disease outbreaks with clinical signs that would typically be attributed to M bovis infection, such as nonresponsive pneumonia or tenosynovitis and arthritis. However, it was not possible to determine whether the calves that had positive results for M bovis via culture, PCR assay, or both had pneumonia attributable to M bovis or other respiratory pathogens. The correlation between nasal shedding and isolation of M bovis from the lower portion of the respiratory tract has varied in previous studies; good²⁰ and poor¹⁰ correlations have been found. The purpose of the present study was not to determine the prevalence of disease attributable to M bovis but to characterize the prevalence of nasal shedding in calves entering backgroundingstocker operations. Further research will be necessary to determine whether calves that shed M bovis at entry are likely to develop disease later in the backgrounding or feeding period or whether calves shedding at entry are an important source of infection to other calves. Circumstantial evidence indicates that calves shedding M bovis can transmit the agent to susceptible calves, resulting in substantial clinical disease.² However, the herds sampled in the present study had, in most instances, no history of a particular problem with disease attributable to M bovis, so it appears that shedding did not necessarily lead to important disease in cattle that were stressed by recent shipment and mixing with calves from a variety of sources. Because treatment of established pneumonia or arthritis-tenosynovitis caused by M bovis is often unrewarding,^2–5 it is important to develop methods to prevent such diseases.