Seroprevalence of Mycoplasma bovis infection in dairy cows in subtropical southern China

State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, CAAS, Lanzhou 730046, People’s Republic of China. Animal Reproduction Institute, Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530005, People’s Republic of China. College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, People’s Republic of China. Guangxi Veterinary Research Institute, Nanning 530001, People’s Republic of China. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, People’s Republic of China.


INTRODUCTION
Mycoplasma bovis is one of major bovine pathogens and is associated with a number of diseases including respiratory diseases and severe otitis, and most commonly implicated in mastitis and pneumonia (Nicholas and Ayling, 2003;Foster et al., 2009;Ball and Nicholas, 2010).M. bovis infection is chronic and nonresponsive to antimicrobial treatment, and it usually co-exists with other bacteria.Therefore, disease outbreaks usually have high morbidity rates, which cause *Corresponding author.E-mail: xingquanzhu1@hotmail.com .Tel: +86 931 8342837.Fax: +86 931 8340977.great economic loss and can be economically devastating to the affected farms, especially in developing countries (Butler et al., 2000;Stipkovits et al., 2000;Gagea et al., 2006;Caswell and Archambault, 2009;Maunsell et al., 2009;Wiggins et al., 2011).However, despite the widespread socioeconomic impact of the pathogen, knowledge on the seroprevalence of M. bovis in China is scarce and there has been only one epidemiological investigation of M. bovis in dairy cows infection in Chongqing, China conducted so far, which revealed a seroprevalence of 20 to 80% (Ran et al., 2010).
Dairy cow is one of the most important farm animals for milk production in Guangxi Zhuang Autonomous Region (GZAR), in subtropical southern China.The objective of

MATERIALS AND METHODS
The study was conducted between June 2009 and March 2010 in 6 main cow-rearing districts of 4 administrative cities in GZAR.These 4 cities are the main cow breeding and milk producing areas in GZAR.Cows were randomly selected and blood samples were collected from tail vein of 455 dairy cows (Table 1).The serum was removed by centrifugation at 4,000 ×g for 10 min and stored at -20°C until testing.Serum samples were tested by the indirect enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Bovine Mycoplasma antibody ELISA kit, Jiahui BioTech, Ltd, Xiamen, China).Positive and negative controls were provided in the kit.Optical density of the samples was read with a photometer (BIO-RAD, Hercules, California, USA).The testing was performed according to the manufacturer's instructions.Basic biometric data on dairy cows including age (records from 211 cows), number of pregnancies (from 207 cows) and history of abortions (from 268 cows) were obtained from the owners by personal interview.

Statistical analysis
Statistical analysis of M. bovis prevalence in dairy cows of different age (in years), with abortion or without abortion, and different numbers of pregnancies were performed by chi-square test using the SPSS software (Statistical Analysis System, Version 11.5, Chicago, Illinois).The differences were considered statistically significant when p < 0.05.

RESULTS
Antibodies to M. bovis were found in 35 out of 455 dairy cows yielding the overall seroprevalence of 7.69%.M. bovis seroprevalence in dairy cows from different geographical locations ranged from 3.3 to 14.89% (Table 1).
The highest seroprevalence of 15% (3/20) was found in 3-year-old dairy cows followed by 11.11% in 4-year-old cows (4/36).No seropositive samples were found in 1 year-old animals and 7 year-old cows (Table 2).No relationship between the seroprevalence and the age could be revealed.
The highest prevalence was found in dairy cows with 5 pregnancies (33.33%), followed by dairy cows having 1 and 3 pregnancies (6.12%).The M. bovis seroprevalence in dairy cows with different numbers of pregnancies ranged from 0 to 33.33%, but there were not statistically significant differences among different groups (p > 0.05).No M. bovis antibodies were found in dairy cows with 0, 6 and 7 pregnancies (Table 3).The seroprevalence of M. bovis in non-abortion dairy cows was 8.33% (22/264).There were 4 cows with abortion, including 2 dairy cows that aborted 3 and 2 times, respectively, but they were all seronegative for M. bovis antibodies.

DISCUSSION
M. bovis is a major cause of calf pneumonia, mastitis and arthritis, and a comprehensive understanding of M. bovis seroprevalence in dairy cows is essential for the control and prevention of M. bovis infection.M. bovis infection have been reported globally, with prevalence of 13 to 23% in Ireland, 25 to 50% in UK, 70% in USA, and 60 to 100% in France (Byrne et al., 2001;Nicholas et al., 2003;Arcangioli et al., 2008).It is estimated that up to 1.9 million cows become infected annually in UK, with 157,000 calves lost due to pneumonia and related diseases (Nicholas and Ayling, 2003).However, there was only one survey of M. bovis seroprevalence in Chongqing, China.
This study indicates that the overall M. bovis seroprevalence in GZAR was lower than that in other countries (Byrne et al., 2001;Nicholas and Ayling, 2003;Arcangioli et al., 2008) and in Chongqing, China (Ran et al., 2010), but close to the lowest level in Ireland (Byrne et al., 2001).However, it was also found that there were particular groups which had high M. bovis seroprevalence, such as those of 3 and 4-year-old as well as those with a history of 1, 3 and 5 pregnancies.Cows of 3 to 4 years old were the main producers of milk.Thus, high M. bovis seroprevalence in these ages will increase the risk of milk contamination with M. bovis.In addition, cows of 3 or 4 years old and those with a history of a few pregnancies were the main part of a herd, therefore, carrier dairy cows are the most likely source for the infection for calves in M. bovis-positive dairy herds.
The seroprevalence of M. bovis in GZAR in dairy cows without abortion was 8.33%, and no M. bovis antibodies were found in aborting dairy cows, including those with high frequency of abortions (2 and 3 times for each).This suggests that no association between bovine abortion and M. bovis infection exists in GZAR and abortions might be caused by factors other than M. bovis infection.
Results of this study indicate that infection of dairy cows with M. bovis was widespread in GZAR.It was reported that healthy dairy cows can be infected with M. bovis by clinically healthy calves or young cows shedding the Mycoplasma, and the pathogen is then very difficult to be eradicated once introduced into a herd, and there are no commercial vaccines for M. bovis at present (Nicholas and Ayling, 2003;Nicholas et al., 2009).Therefore, integrated strategies and measures should be executed to prevent and control M. bovis infection.

Table 1 .
Seroprevalence of M. bovis infection in dairy cows in different cities, in Guangxi ZhuangAutonomous Region (GZAR), China.

Table 2 .
Seroprevalence of M. bovis infection in dairy cows of different ages in GuangxiZhuang Autonomous Region (GZAR), China.
this study was to investigate the seroprevalence of M. bovis infection in GZAR.The results would provide baseline data for the implementation of effective strategies for the control of M. bovis infection in dairy cows in China.

Table 3 .
Seroprevalence of M. bovis infection in dairy cows with different numbers of pregnancies in Guangxi Zhuang Autonomous Region (GZAR), China.