Trop Anim Health Prod DOI 10.1007/s11250-017-1271-2
REGULAR ARTICLES
Characteristics of Aerococcus viridans isolated from bovine subclinical mastitis and its effect on milk SCC, yield, and composition Meng Sun 1 & Jian Gao 1 & Tariq Ali 1 & Dan Yu 1 & Shiyao Zhang 1 & Saeed U. Khan 2 & Séamus Fanning 3 & Bo Han 1
Received: 1 April 2016 / Accepted: 14 March 2017 # Springer Science+Business Media Dordrecht 2017
Abstract Aerococcus viridians (A. viridans), an environmental Gram-positive bacterium, has been documented to be associated with bovine mastitis. However, its exact role in bovine mastitis and the changes it brings about in milk characteristics are not yet known. The objectives of the current study were to describe the antibiotic resistance of A. viridans from bovine mastitis as well as the correlation between existence of this pathogen in udders and the somatic cell counts (SCC), daily milk yield, and composition of individual cow. Oneyear sampling for subclinical mastitis composite milk was conducted based on monthly DHI data from September 2013 to August 2014, in a commercial herd located in Beijing, China. All samples were cultured and pathogens were identified using microbiology method. A. viridians isolates were further identified by API identification system and 16S ribosomal RNA (rRNA) sequencing method. Kirby-Bauer disk diffusion method was used to test the antibiotic resistance of A. viridians against kinds of antimicrobial substance. SCC, milk yield, and milk composition data were from monthly Dairy Herd Improvement (DHI) results. Results showed that
a total of 279 (16.67%) A. viridans isolates were identified from among 1674 bacterial isolates cultured from milk samples with high SCC. The incidence of mastitis caused by A. viridans was the highest (48–53%) during the summer season. Majority of the isolates were susceptible to most of antimicrobial compounds tested, especially to β-lactams, but were found to be resistant (50–90%) to aminoglycosides, sulfonamides, and tetracycline. The average SCC of the A. viridans infected cows was significantly higher (1000.0 × 103 cells/mL) (P < 0.01) as compared to healthy cows (72.4 × 103 cells/mL) and daily milk yield decreased (P > 0.05) by 1.86 kg/day. Reductions were also observed in fat content (P > 0.05), lactose (P < 0.01), and total solids (P > 0.05), whereas protein content increased significantly (P < 0.01) in milk samples of cows infected with A. viridans. The results of this study suggest that A. viridans could be considered as an emerging aetiological agent of bovine subclinical mastitis wherein it exerts an effect on SCC, milk yield, and composition. Keywords Aerococcus viridans . Antibiotic susceptibility . Daily milk yield . Milk composition . Subclinical mastitis
Meng Sun and Jian Gao equally contributed to this work. * Bo Han
[email protected] 1
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, 100193 Beijing, People’s Republic of China
2
Department of Animal Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
3
UCD-Centre for Food Safety, School of Public Health, Physiotherapy & Sports Science, University College Dublin, Belfield, Dublin 4, Ireland
Introduction Bovine mastitis is still a very common and costly disease to the dairy industry throughout much of the world. Mastitis also affects animal welfare (Bradley 2002) and is a frequent reason that cows are permanently removed from dairy production. Bacterial infection is the main reason for mastitis. Aerococcus viridans, belongs to streptococcaceae family, and a saprophytic bacterium can be found in the environment. This microorganism closely resembles streptococci and can easily be misdiagnosed due to their closely related properties
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(Rasmussen 2013). Clinically, this bacterium is associated with some of the diseases of humans and animals (Chen et al. 2012; Zhou et al. 2014). In the veterinary context, it has been reported to cause infections in lobsters (Stebbing et al. 2012), tilapia fish (Ke et al. 2012), swine (Martín et al. 2007), and cattle (Guccione et al. 2013). More recently, A. viridans has been reported to be associated with bovine mastitis in East Asia (Liu et al. 2015; Saishu et al. 2015). Mastitis could be categorized into clinical and subclinical forms. In clinical mastitis, symptoms could be observed in milk, udder or cow (Fogsgaard et al. 2015). While, subclinical mastitis is characterized by an increase in somatic cell counts (SCC) without any visual abnormality in the udder or milk. In dairy cows, a SCC higher than 200,000 cells/mL indicates subclinical mastitis (National Mastitis Council 2001; Schukken et al. 2003). Economic losses due to subclinical mastitis are much greater since higher prevalence (Halasa et al. 2007). Aerococcus viridans is emerging as an aetiological agent in cases of bovine mastitis. It has been previously reported to be associated with bovine mastitis (Zadoks et al. 2004; Špaková et al. 2012; Liu et al. 2015; Saishu et al. 2015); however, its precise role in bovine mastitis and the resulting changes in milk SCC and milk composition have not been investigated. This study was mainly aimed to describe the antibiotic resistance of A. viridans isolated from bovine mastitis, as well as correlation between existence of this pathogen within udders and the SCC, daily milk yield, and composition of individual cow. To the best of our knowledge, this is the first comprehensive report highlighting the role of A. viridans in subclinical mastitis and describing its effect on the mastitis-related indicators.
The farm was visited on a monthly basis for 1 year from September 2013 to August 2014. Each farm visit was conducted at the following day after monthly DHI data of this farm was supplied by the herdsmen. If SCC of the individual cow was above 200,000 of cells/mL, it is considered as a subclinical mastitis cow. The cow was filtrated and ID of cow listed by researchers for sampling in the following day. A cow which had been sampled was excluded from the future sampling. Thus, all samples were from different individual cows. In order not to interfere with common operation in milking parlors, researchers were unable to sample all cows involved in the sampling list. In general, three or four researchers themselves could collect more or less 100 of cow composite samples per visit. Samples were collected before milking with discarding three to five squirt of milk. Five milliliters of milk were collected aseptically and equally from different quarters of an individual cow according to the standard method recommended by National Mastitis Council (2001) and mixed together as about 20 ml of composite samples. Samples were transported under 4 °C a to microbiological laboratory on the same day and cultured. Due to the possibility of false positive results, milk samples from cows in the first and last week of lactation were excluded (Radostits et al. 2007). Bulk milk SCC of the herd was normally kept in 300,000 to 400,000 cells/mL. Incidence rate of clinical mastitis was 40 to 50 cases per 100 cows per year. Antibiotics were used to treat only clinical cases. The animals did not receive any specific treatment before the study. In addition, outbreak of other diseases was not found during the period of farm visit.
Isolation and identification of A. viridans
Materials and methods Dairy herds and sampling A total of 1145 milk samples were collected from a commercial dairy herd composed of 2150 Holstein milking cows, located in Beijing. All cows were feed with total mixed ration and milked automatically in a double-40 parallel milking parlor. The cows were kept in free stall and laid on sand beddings. The herd was chosen because A. viridans had been found in bulk tank milk by real-time PCR test and isolated for several times from clinical and subclinical mastitis quarters. In addition, the herd was involved in the national DHI program so monthly DHI data of milking cows could be supplied for analysis. The SCC of individual cows was performed using a somatic cell counter (CombiFoss FT™, Foss Electric, Hillerød, Denmark). Researchers visited the herd per month for the collection of milk samples and other related parameters.
Ten microliters of milk sample was inoculated onto trypticase soya agar (Sigma, Shanghai, China) containing 5% sheep blood and then the plates were incubated at 37 °C for 24 h. Bacterial isolates other than A. viridans were identified by microbial morphology and biological chemistry test following the scheme prescribed by the National Mastitis Council (2001). Initial screening of suspected A. viridans colonies was done based on their colonial morphology, hemolysis reaction, and Gram stain (Liu et al. 2015). Further identification of A. viridans was performed using commercially available kits of API Rapid 20 strep (bioMérieux, Marcy I’Etoile, France). The identification of A. viridans was further confirmed by PCR amplification of a 750-bp fragment of the 16S rRNA using universal primers (Frank et al. 2008), followed by commercial sequencing (Sunbiotech, Beijing, China) and BLAST analysis. A. viridans species were identified at ≥98% homology level.
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Antimicrobial susceptibility test Antibiotic susceptibility of A. viridans isolates against 17 different antimicrobial agents was performed using the Kirby-Bauer disk diffusion method following guidelines of the Clinical Laboratory Standard Institute (CLSI 2013). In brief, one bacterial colony was suspended in 2 mL Brain Heart Infusion (Difco™, Shanghai, China) broth and grown overnight in shaking incubator at 37 °C. Three suspensions were made and adjusted to approximately 1.5 × 108 CFU/mL using the McFarland standard. The three suspensions were inoculated using sterile cotton swabs onto Muller-Hinton agar (Oxoid, Shanghai, China) and incubated at 37 °C for 24 h. The antibiotic disks (BD BBL, Sparks, MD, USA) and their concentrations are listed in Table 2. The results for antibiotic susceptibility were interpreted using previously described breakpoints for the resistance (Martín et al. 2007; CLSI 2013). Existence of A. viridans in cow composite milk samples and elevated SCC Milk samples with elevated SCC counts (> 200,000 cells/mL) were spread onto a blood agar plate. The cow was considered as A. viridans infected animal if 10 or more colony-forming units (CFU) of A. viridans were observed after 24-h incubation at 37 °C (Nam et al. 2010). Though it was hard to define a Table 1 Incidence of Aerococcus viridans among major bacteria recovered from cases of bovine subclinical mastitis Type of bacteria
No. of isolates (n = 1674)
Frequency (%)
Coagulase-negative staphylococcus Aerococcus viridans Staphylococcus aureus Escherichia coli Environmental streptococci Trueperella pyogenes Corynebacterium xerosis Enterobacter species Bacillus subtilis Streptococcus agalactiae Streptococcus uberis Klebsiella
572 279 200 172 135 91 50 45 34 25 25 13
34.17 16.67 11.95 10.27 8.06 5.44 2.99 2.69 2.03 1.49 1.49 0.78
13 6 6 4 2 2
0.78 0.36 0.36 0.24 0.12 0.12
Corynebacterium bovis Enterococcus faecalis Serratia Acinetobacter Proteus species Listeria monocytogenes
composite sample as contaminated using the criteria described by previous study (Parker et al. 2008), we still considered the sample as contaminated when three or more microbial species were observed on the same plate. If samples with elevated SCC were culture negative, it was considered to come from healthy cows. Of samples from healthy cows, 200 were selected for comparing with SCC data of samples containing A. viridans. Milk yield and milk composition analysis Data regarding daily milk yield and milk composition including fat (%), lactose (%), protein (%), and total solid (%) were analyzed by DHI testing laboratory (CombiFoss FT™; Foss Electric, Hillerød, Denmark). The DHI data sheets of 200 randomly selected healthy animals and cows suffered from subclinical mastitis for 12 consecutive months were obtained from the farm’s owner. Comparison was made between the healthy cows and cows infected with A. viridans and these data were statistically analyzed. Statistical analysis Study animals were divided into two groups. The mean lactation of cows in the healthy group was 3.05 ± 1.12 while 3.09 ± 1.04 in the A. viridians group. The mean milking days of cows in the healthy group was 191 ± 66 while 182 ± 71 in the A. viridians group. The SCC data were normalized to the log10 scale (Ali and Shook 1980) and then statistical analyses were performed using the SPSS software v20.0 (SPSS Inc., Chicago, IL). Means and standard deviations of the parameters were calculated. Independent sample t test were used to compare the data. Differences were considered statistically significant or highly significant when P values were
