Post-weaning management strategies and impacts on ewe subclinical mastitis and antimicrobial susceptibility

© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2021.5:S80–S85 https://doi.org/10.1093/tas/txab180


INTRODUCTION
Mastitis, defined as the inflammation of mammary tissue, often results from microbial infection (Kahn and Line, 2010) and is prevalent from parturition to weaning. Subclinical mastitis is prevalent (Ahmad et al., 1992a), although on-farm diagnosis is difficult, especially in non-dairy animals, and bacterial culture remains the standard diagnostic tool. Through managing environmental pathogens, animal and facility management practices may reduce the prevalence of subclinical mastitis. For example, withholding feed from ewes for 48 h following weaning with an additional 2 wk of low-energy, low-protein feed is recommended (American Sheep Industry Association Inc., 2015). The use of antimicrobials to control mastitis and other diseases in the livestock industry (Sawant et al., 2005;Raymond et al., 2006) raises concerns of antibiotic resistant strains affecting human consumers (Skovgaard, 2007). Studies evaluating antimicrobial resistance in dairy sheep report variability in resistance, though bacterial strains most commonly display resistance to penicillin and ampicillin (Concepción Porrero et al., 2012;Azara et al., 2017).
Therefore, the objectives of this study were to quantify the prevalence and etiological agents of subclinical mastitis at weaning, analyze the antimicrobial susceptibility in ewe milk, and determine the effectiveness of weaning treatments to mitigate subclinical mastitis. We hypothesized that most ewes would present subclinical mastitis around weaning and antimicrobial resistance of isolates would be low in the absence of prolonged treatment with antimicrobials.

MATERIALS AND METHODS
The experimental protocol was approved by the University of Wyoming Institutional Animal Care and Use Committee (20200214HC00408-01).

Animals and Management
Multiparous commercial Rambouillet ewes (n = 22) were managed by standard Laramie Research and Extension Center practices and lambed between March and April. Prior to weaning, ewes were assigned to 1 of 3 treatment groups: 1) intramuscular injection of penicillin (PENN; n = 8; Durvet; penicillin G procaine injectable suspension; 300,000 units mL −1 ; 1 mL per 45 kg BW) at weaning, 2) restricted feed access 48 h prior to and 72 h post-weaning (FAST; n = 6), and 3) a combination of penicillin at weaning and restricted feed access (COMBO; n = 8). For FAST and COMBO rations, ewes consumed 1.57% to 1.72% BW grass hay per day, whereas PENN continued to consume 2.85% and 2.47% BW grass and alfalfa hays per day, respectively.

Milk Collection
Milk samples were collected at weaning (68.31 ± 5.47 d) and post-weaning (71.31 ± 5.47 d). Teats were scrubbed with 70% ethanol and the first streams of residual milk were discarded. Approximately 20 mL of milk was obtained from the two udder halves. All aliquots were frozen at −80 °C until sample preparation.

Microbial Culturing and Identification
Thawed milk samples were streaked onto plates containing microbiological media (MacConkey agar; trypticase soy agar (TSA); and TSA + 5% sheep blood agar). Plates were incubated (37 °C for 24 h) and those that exhibited no growth were incubated an additional 24 h. Colony counts and morphologies were recorded, and culture-positive samples were considered to have subclinical mastitis (Dohoo et al., 2011) and then were sub-cultured for isolation and used to make a preserved freezer stock.
Cultures were re-activated on TSA plates for identification via matrix-assisted laser desorption/ ionization spectrometry (MALDI-TOF MS). The extended direct colony transfer method was used by transferring colonies onto a steel-target plate where 70% formic acid (1 µL) and HCCA matrix (1 µL; α-cyano-4-hydroxycinnamic acid) were added to each well. Bruker Biotyper RTC software (Version 3.1) was used for MALDI-Biotyping (Proteomics and Metabolomics Facility; Colorado State University; Fort Collins, CO).

Antimicrobial Susceptibility Testing
Bacteria with a high-quality score for a MALDI-TOF MS identification match were subjected to antimicrobial susceptibility testing. After re-activation of cultures using above methods, colonies were added to demineralized water to achieve a 0.5 McFarland standard before 10 µL of suspension (30 µL for staphylococci) was added to Mueller-Hinton broth. The antibiotic plate (Sensititre Vet Mastitis CMV1AMAF; Thermo Scientific; Waltham, MA) was inoculated with 50 µL per well of suspension to test isolates against ampicillin, penicillin, erythromycin, oxacillin +2% NaCl, pirlamycin, penicillin/novobiocin, tetracycline, cephalothin, ceftiofur, and sulphadimethoxine. The plate was analyzed via a Sensititre Vizion instrument.

Data Analyses
Subclinical mastitis prevalence and MALDI-TOF MS identifications were analyzed within and across weaning treatment and day. Binomial
Translate basic science to industry innovation proportions and 95% confidence intervals of taxa identified within culture-positive samples were estimated using the binom package of R (Dorai-Raj, 2014; RStudio Team, 2020). The survival package of R (Therneau, 2021) was used for a survival analysis of isolates based on penicillin usage at weaning (PENN+ = PENN or COMBO; PENN− = FAST), where antimicrobial concentrations present in plate wells were used as "time" and "event" was inhibition of bacterial growth.

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Graduate competition: ewe mastitis and antimicrobial susceptibility Translate basic science to industry innovation
Upon culturing and confirmation, 17 unique bacterial species were identified as belonging to the genera Bacillus, Brachybacterium, Mannheimia, Moraxella, Staphylococcus, and Streptococcus (Tables 1 and 2). Many of these bacteria are common isolates of subclinical mastitis cases in extensive/ semi-extensive systems (Bacillus spp., Staphylococcus spp., Mannheimia haemolytica, Streptococcus spp.; Arsenault et al., 2008;Knuth et al., 2019) and intensive systems (Staphylococci spp. and Streptococcus spp.; Ahmad et al., 1992b). Although these preliminary data do not report statistical differences in prophylactic management strategies, further research is warranted to identify efficacy of weaning treatments to reduce subclinical mastitis.

IMPLICATIONS
Subclinical mastitis is likely more prevalent than previously thought, and production impacts on ewe productivity and animal welfare are not well understood in wool-type ewes. The present data are preliminary results of a broader investigation of lambing and weaning management practices on subclinical mastitis and the milk microbiome. These data show many potentially pathogenic bacteria isolated from subclinically infected ewes, where some isolates showed resistance to antimicrobial agents that are not extensively administered. Future research is warranted to judiciously employ antimicrobial agents in the livestock industry and ensure animal health while maximizing efficient and effective production.

ACKNOWLEDGMENTS
Funding for this research was provided by the National Sheep Industry Improvement Center.
Conflict of interest statement. Mention of commercial products was solely stated to provide specific information and does not imply recommendation. The authors confirm that the research was conducted in the absence of commercial or financial relationships that could have influenced the outcome of the study.