History and Physical Examination Findings

Herein we reported a mastitis outbreak in a lamb-producing farm, causing a severe decline in lamb performance. Ewes were affected by clinical and subclinical udder infections. Milk samples were aseptically collected and cultured for microbial isolation. Methicillin-resistant Staphylococcus aureus (MRSA) was identified as the causative agent of mastitis in an affected ewe. Considering that MRSA is 1 of the most common agents associated with nosocomial infections worldwide, ranking second in the number of deaths caused by antimicrobial drug-resistant bacteria in the US,¹ we decided to further investigate the strain.

Importantly, some MRSA strains are developing resistance to virtually all β-lactams, the most widely used class of antimicrobials, including penicillin, methicillin, and carbapenems.²

This is the first report on the occurrence of clinical mastitis caused by MRSA sequence type (ST)-398 in a sheep herd.

Diagnostic Findings and Interpretation

We performed an investigation on a mastitis outbreak in a meat-producing sheep herd, comprised of 300 ewes and 85 replacement lambs in Ohio. It was a semiextensive production system based solely on alfalfa stands. Ten ewes affected by clinical mastitis and also having poor lamb performance were physically examined. One sheep in particular showed fever, reactive mammary lymph nodes, thinness, muscle weakness, and chronic mastitis in both udder halves, 1 of them with a draining pus fistula.

A sample of purulent discharge was collected from this ewe with a sterile swab and stored in transport media (Stuart Transport Medium; Thermo Scientific). The sample was streaked onto blood agar 5% (Becton Dickinson BD) and incubated at 37 °C for 12 hours. Five typical staphylococci colonies with marked β-hemolysis were streaked onto mannitol salt agar (Becton Dickinson BD) and oxacillin screen agar (Becton Dickinson BD) and incubated at 37 °C for 24 hours. Bacterial growth was observed in both agars. Catalase-positive isolates were tested for coagulase production using a commercial kit (Coagulase Test; Sigma-Aldrich).

Conventional microbiological procedures used for the isolation and identification of S aureus in the clinical samples are shown in Figure 1.

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Conventional microbiological procedures for the isolation and identification of Staphylococcus aureus isolates. A—Mannitol salt agar plate. B—Oxacillin-resistant screening agar plates (blue colonies are positive for oxacillin resistance). C—Coagulase test (fibrin clot formation is positive for coagulase). + = Positive result. − = Negative result.

Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.05.0137

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DNA from the isolates was extracted using a commercial kit (DNeasy Blood and Tissue; QIAGEN) according to the manufacturer’s protocol. Duplex PCR assays targeting nuc and mecA genes for S aureus species identification and methicillin resistance confirmation, respectively, were performed as detailed in Supplementary Material S1. Shortly, a DNA template (50 ng) was added to a 24-µL master mix prepared using a commercial kit (Illustra PuReTaq Ready-To-Go PCR Beads; GE Healthcare).

Multilocus sequence typing (MLST) was performed using an established global protocol available at Public Databases for Molecular Typing and Microbial Genome Diversity/PubMLST (https://pubmlst.org/organisms/staphylococcus-aureus/primers).³ Information on the sequencing primers targeting the 7 housekeeping genes carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyle coenzyme A acetyltransferase (yqiL) is provided in Supplementary Table S1. Allelic profiles and the respective STs were determined using the PubMLST database (http://pubmlst.org/).

The investigated isolates harbored both nuc and mecA genes. Considering both phenotypic and molecular findings, the isolates were considered true MRSA. The MLST analysis revealed that the MRSA isolates from the sheep herd belonged to ST398, with the following housekeeping gene allelic profile: 3-35-19-2-20-26-39.

Treatment and Outcome

Due to the severe clinical conditions of the ewe and lamb, they were euthanized according to the AVMA guidelines for animal euthanasia.⁴ The Ohio State IACUC approved the protocols used in this study.

Necropsy revealed adhesions of the right lung and chest wall; fibrin deposits on the chest wall; consolidated and firm apical lung lobe; consolidated and firm middle lung lobe; pale musculature and carcass appearance (Figure 2); prominent mesenteric lymph nodes, color normal; pale liver; no evidence of nematodes (Haemonchus) in the abomasum; pale blood appearance; and normal gastrointestinal contents. Biochemical tests revealed that the lamb had mineral deficiency and was more susceptible to infections and parasitic diseases, resulting in unsatisfactory growth.

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Macroscopic lesions and adhesions of the right lung and chest wall from euthanized ewe and lamb affected by severe clinical mastitis. A—Necropsy of ewe shows apical lung lobe consolidated and firm; middle lung lobe consolidated and firm. B—Necropsy of lamb shows adhesions of right lung and chest wall and fibrin deposits on chest wall.

Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.05.0137

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Comments

To the best of our knowledge, this is the first report on ovine clinical mastitis caused by MRSA ST398, a zoonotic staphylococcal lineage associated with livestock-associated MRSA (LA-MRSA).⁵ Staphylococci organisms are the most common causative agents of mastitis in ruminants. Although the clinical form of mastitis represents a small percentage of mastitis cases in small ruminants, usually less than 5%, it is the form of mastitis that the producers are most aware of.⁶ Mastitis has a large impact on the economy as well as on animal welfare in sheep production, but the zoonotic potential of mastitis-causing agents has not been properly acknowledged despite their infectious potential being reported by dairy farmers.⁵

MRSA is a leading pathogen in human medicine and is normally detected in clinically healthy animals from different species.¹ In the past 15 years, the transmission of LA-MRSA from animals to humans has gained special attention, and reports of infections, especially in livestock workers, have been increasingly documented.¹ Recent epidemiological studies^1,5 demonstrated that food animals could serve as reservoirs of LA-MRSA that can cause human infections. Therefore, information on MRSA in animals is important considering its potential to cause occupational diseases in farmers, handlers, and veterinarians.²

Although MRSA strains are typically β-lactam–resistant bacteria, they are considered multidrug-resistant strains harboring other genetic determinants conferring resistance to a range of different antimicrobial classes, such as aminoglycosides, fluoroquinolones, and tetracyclines.² Furthermore, LA-MRSA ST398 frequently harbors genes related to the immune evasion cluster of humans and the invasion of the mammary gland during cow mastitis,⁶ which may explain the severe mastitis condition. Moreover, the clinical case reported herein highlights the remarkable ability of MRSA to adapt to different hosts, with serious implications for public health.

Therefore, the global threat imposed by antimicrobial resistance and the increasing debate on the importance of livestock as potential sources of antimicrobial-resistant strains to humans have led to improved surveillance of antimicrobial-resistant pathogens at the farm level. Our investigation reveals that sheep can serve as reservoirs of MRSA ST398, which poses a public health concern due to the risk of occupational staphylococcal infections in humans (LA-MRSA). These findings warrant further investigations on the epidemiology of MRSA in sheep herds as factors associated with the maintenance and transmission of MRSA in sheep herds remain uncertain.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

Funding

The authors are thankful to the Brazilian National Council for Scientific and Technological Development (CNPq; proc. CsF 200880/2012-8) for providing grants and scholarships to Dr. Moura in the scope of the Science without Borders program. CJBO holds a Research Productivity Fellowship (Level 1-D; CNPq, proc. 3136678/2020-0 also provided by CNPq.

ORCID

M. M. S. Saraiva https://orcid.org/0000-0003-1875-4495

D. T. Stipp https://orcid.org/0000-0002-9577-2446

W. A. Gebreyes https://orcid.org/0000-0003-1885-3952

C. J. B. Oliveira https://orcid.org/0000-0002-7761-0697