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Evaluation of Mollicutes Microorganisms in Respiratory Disease of Cattle and Their Relationship to Clinical Signs

Background Bovine respiratory disease (BRD) is an important problem in cattle production that is responsible for economic losses in dairy herds. Mycoplasma spp. are described as an important etiological agent of BRD. Hypothesis To evaluate the occurrence of the most important mycoplasmas in the lower respiratory tract of healthy and BRD cattle in relationship to clinical signs of BRD. Animals Sixty young dairy cattle were classified as healthy (n = 32) or cattle showing clinical signs of BRD (n = 28). Methods Tracheal lavage samples were collected and added to tubes containing Hayflick media. Mycoplasma spp. were identified by the presence of “fried egg” like colonies, biochemical tests and polymerase chain reaction (PCR). Occurrence of Mollicutes, M. bovis, M. mycoides subsp. mycoides SC and M. dispar was evaluated. The association between clinical signs of BRD and the presence of Mycoplasma spp. also was evaluated. Results Colonies were obtained from a 1‐year‐old BRD calf only. However, species identification was not possible. Mollicutes (P = .035) and M. dispar (P = .036) were more common in BRD cattle. The relationship between Mollicutes and crackle (P = .057) was not significant. M. dispar was associated to tachypnea (P = .045) and mixed dyspnea (P = .003). Relationships to heart rate (P = .062) and crackle (P = .062) were not significant. Conclusions and clinical importance The results confirmed the importance of mycoplasma as an etiologic agent of BRD and suggested M. dispar as part of the respiratory microbiota and its possible role in the development of BRD.

B ovine respiratory disease (BRD) is an important problem in cattle production that remains responsible for economic losses in dairy and feedlot herds because of high morbidity and mortality rates. [1][2][3] It is observed in young calves, 4 particularly between 2 and 6 weeks of age. 5,6 In addition, BRD negatively impacts growth, reproductive performance, and longevity. 5,7 Mycoplasma spp. belong to the Mollicutes class, and they are described as important etiological agents of BRD. 3 mycoides small colony is the etiological agent of contagious bovine pleuropneumonia, 10 and it is considered the most pathogenic mycoplasma. Mycoplasma mycoides subsp. mycoides SC has never been detected in Brazilian cattle, although its detection in the external auditory meatus of clinically healthy goats was described elsewhere. 11 M. bovis is an opportunistic bacterium considered part of the bovine respiratory tract microbiota. 12 After stressful situations, M. bovis becomes pathogenic and clinical signs of BRD are observed, especially in young calves. 13 M. dispar was first isolated from pneumonic lungs of cattle, 14 and it has been described as a potential pathogen associated with BRD. 15,16 Considering the importance of Mycoplasma spp. in the development of BRD, the aim of our study was to evaluate the occurrence of the most important mycoplasma species in the lower respiratory tract of healthy and sick Brazilian cattle in relationship to clinical signs of BRD.

Materials and Methods
The study was conducted at the Internal Medicine Department, School of Veterinary Medicine and Animal Science, University of São Paulo and at the Laboratory of Mycoplasmas, Institute of Biological Sciences, University of São Paulo, Brazil. All procedures were carried out in agreement with the guidelines of Ethical Principles in Animal Research adopted by the Ethic Committee on the Use of Animals of the School of Veterinary Medicine and Animal Science of University of São Paulo.
Sixty young dairy cattle were randomly selected and enrolled in the study. Fifty-eight cattle were from 10 farms located in the state of São Paulo, Brazil. Calves were immediately separated from their mothers after birth. They received colostrum and milk by farm employees, and after weaning, they received a pasture and barley-based diet and mineral salt. Two cattle were presented at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science, University of São Paulo.

Case Definition
Bovine respiratory disease was diagnosed when the animal showed ≥2 of the following clinical signs: mucopurulent or purulent nasal discharge, cough, rectal temperature >39.5°C, respiratory rate >40 breaths/min, and increased cranioventral lung sounds or crackle. 6,17,18 The limits of the lung field were 12°i ntercostal space at iliac line and 11°intercostal space at sciatic line. Two experienced veterinarians on our research team performed the physical examinations in all cattle. Animals were allocated in 2 groups: healthy (n = 28) and BRD cattle (n = 32).

Sample Collection
The distal part of the neck was shaved and decontaminated with 70% alcohol and iodopovidone. Twenty milliliters of sterile saline 0.9% was instilled with a 16 9 40 mm needle and up to 5 mL was recovered. Samples were added to tubes containing Hayflick media and transported on ice to the laboratory.

Cultivation of Mycoplasma spp.
Clinical samples were diluted (10 0 , 10 À1 , 10 À2 , 10 À3 ) in phosphate-buffer saline (PBS). Mycoplasma spp. isolation was performed by plating 100 lL of each dilution in Hyflick media growth plates and adding 200 lL of each dilution in 1800 lL of liquid media containing Hyflick media. 19 Plates and liquid media were incubated at 37°C for 21 days and evaluated on a daily basis. Plates containing "fried-egg" colonies and glucose fermentation with or without arginine hydrolysis were considered positive. Liquid media containing glucose fermentation with or without arginine hydrolysis and absence of turbidity were considered positive.

Molecular Detection
Molecular investigation of Mycoplasma spp was performed using DNA extraction according to a previously described procedure. 20 Polymerase chain reaction was performed to investigate the presence of Mollicutes class bacteria. 21 Positive samples were used to detect M. bovis, 22 M. dispar, 23 and MmmSC. 24

Statistical Methods
Descriptive analysis was performed to determine absolute and relative frequencies. The occurrence of Mycoplasma spp. was considered the dependent variable. Health status and clinical signs were considered the independent variables. The association between the presence of Mycoplasma spp. and health status and clinical signs of BRD was compared by applying the Pearson's chi-square test or Fisher's exact test using a 95% confidence interval. Clinical data were analyzed by the Statistical Package for Social Sciences 19.0. Variables with P < .05 were considered significant.
Colonies were obtained from 1 BRD calf only. However, species identification was not possible because of the low quality of the sample.
Polymerase chain reaction was performed to detect Mollicutes, M. dispar, M. bovis, and MmmSC. Mollicutes were increased in BRD cattle (68%) compared to healthy cattle (41%; P = .035; Table 2). Specific PCR tests to detect M. dispar, M. bovis, and MmmSC were performed in 25% (8 of 32) and 64% (18 of 28) of samples from healthy and BRD groups, respectively, because of the low quality of the other samples. M. dispar was increased in BRD group (61%) compared to the healthy group (12.5%; P = .036), M. bovis was detected in BRD animals only (5%), and no difference between groups was noted (P = .497;  The association between the bacteria detected and clinical signs of BRD was evaluated. (Table 3). With regard to M. dispar, tachypnea was more common in positive animals (66.7%) as compared to negative animals (21.4%; P = .045). Mixed dyspnea (inspiratory and expiratory dyspnea) was more common in the positive group (66.7%) compared to the negative group (7%; P = .003). No significant association between clinical signs and M. dispar was observed (Table 4).

Discussion
To better understand the importance of Mycoplasma spp. in BRD, we evaluated the occurrence of Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma mycoides subsp. mycoides SC. in tracheal wash samples of healthy and BRD cattle in association with clinical signs of BRD. Our results indicated that M. dispar was common in BRD animals, confirming its importance as a pathogen of BRD. Association between Mollicutes and some clinical signs of respiratory diseases was detected.
Colonies were obtained from 1 sample only, unlike the high isolation rates of Mycoplasma spp. described elsewhere. 5,15,25,26 Mycoplasma spp. are well-known as fastidious and slow-growing bacteria for which isolation takes an extended time. 27 Polymerase chain reaction is a quick and sensitive test that can detect nucleic acid from only 1 microorganism when it is used to detect Mollicutes. 21 Often, culture negative samples are positive for molecular detection, as observed in our study.
Mollicutes was increased in the BRD group (P = .035). Similarly, Mollicutes have been reported frequently in more BRD calves (90.96%; 53%) compared to healthy calves (52.05%; 23%). 27, 28 Mollicutes are well characterized as part of the bovine respiratory tract microbiota 27,29 but several species have been described as etiologic agents of respiratory diseases. 30 M. dispar was increased in BRD cattle (P = .036). Our data are in agreement with a previous study, 31 which also described the increased occurrence of M. dispar in both healthy and BRD groups, especially in the latter group. Two other studies also detected a high occurrence of M. dispar in BRD cattle compared to healthy cattle. 15,27 Mycoplasma mycoides subsp. mycoides SC is the etiologic agent of contagious bovine pleuropneumonia, and it is considered the most important mycoplasma species related to BRD. 10 In our study, this species was not detected, and this result is in accordance with other Brazilian studies. 27,31 M. bovis is another important mycoplasma related to BRD. 29,32 In our study, however, this bacterium was detected in 1 BRD calf only. Similar results were obtained in another study. 33 Undetermined mycoplasma species were observed in both groups. Ureaplasma diversum, Acholeplasma spp. and other mycoplasma species such as M. bovirhinis, M. alkalensis, and M. arginini have been detected in the bovine respiratory tract. 15,16,[25][26][27] The genus Mycoplasma has several species, and culture-independent techniques are indispensable to determine all species present in the respiratory tract.
Regarding clinical signs of BRD, our data identified Mollicutes and M. dispar associated with respiratory problems. Our results establish association but not necessarily causation. Another study found that the presence of a clinical sign of BRD (stony dull sound on percussion of the thorax) was related to the absence of Mollicutes. 31 Regarding M. dispar, our data indicated an association between this bacterium and tachypnea and mixed dyspnea. In an experimental infection with M. dispar in calves, most calves showed no clinical signs of BRD. 34 However, only 1 calf showed persistent nonproductive cough and dyspnea, besides increased respiratory rate and fever. Recently, another study indicated that coarse crackles and whistling were associated with the absence of M. dispar. 31 Mycoplasma dispar is regularly isolated from bovine pneumonic lungs, but its presence has been associated with mild infection. 30,34 Discrepancies in results allow researchers to continue studying these microorganisms to better understand the importance of mycoplasmas in the development of clinical signs of BRD. In addition, it is important to note that other microorganisms could contribute to BRD.

Conclusion
Our study confirmed the importance of mycoplasmas as etiologic agents of BRD. Although M. dispar has been detected in healthy cattle, the increased occurrence of this bacterium and the detection of M. bovis in BRD calves confirm their roles in the pathogenesis of BRD. The increased frequency of undetermined mycoplasma species in samples indicates the complexity of the respiratory tract microbiome and the possible role of other mycoplasmas in BRD. This new information about the association between some clinical signs of BRD and Mycoplasma spp. infection will be useful in the presumptive identification of the microorganisms involved in BRD infection.