We describe the case of a three-year old Holstein dairy cow (1428) who presented with a naturally-occurring, subclinical mastitis infection in her left hindquarter, approximately two months into her first lactation. Milk samples from cows in our research herd are periodically monitored for bacterial growth and changes in SCC (Somatic Cell Count) to monitor animal and udder health. Additional samples are collected if naturally occurring mastitis is suspected or for various scientific uses. Cow 1428 was born and raised on the USDA campus within the Holstein research herd. Mastitis was observed at a routine daily milking and culture of the sample was performed. Milk from quarters of interest was aseptically collected, by hand milking, and SCC sample values were determined by Dairy Lab Services (IA, USA). For bacterial counts, aseptically collected milk samples were plated on Trypticase Soy Agar with 5% sheep blood plates (BD Biosciences, CA, USA Cat. No.221261), and incubated overnight at 37 °C, prior to colony counting. An isolated colony was typed by the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) and identified as S. aureus. The S. aureus strain was sequenced and designated as SA1428 [24]. The infection remained subclinical, with no identifiable drop in milk yield, no visual signs of inflammation including teat hardening, redness, or milk chunkiness, but was continuously identifiable by moderately increased SCC and bacterial culture. Multiple SCC and bacterial counts were determined over the course of several months. At the initial detection of the infection SCC in the infected quarter were 3.8 × 106 cells/ mL with bacteria counts > 3000 cfu/mL. Other quarters had no detectable bacteria and SCC below 7.5 × 104 cells/ mL. Cow 1428 was not isolated from the herd, however, no other cows became naturally infected with the novel S. aureus pathogen to our knowledge.
Cow 1428 was treated with antibiotics, daily for five days, with cephapirin sodium (ToDAY, Boehringer Ingelheim, MO, USA) twice daily, and additionally pirlimycin hydrochloride (PIRSUE, Zoetis, NJ, USA) once daily. In our herd, this treatment has been successful at clearing experimentally induced S. aureus infections (Newbould 305 strain). When antibiotics did not clear the infection (as confirmed by bacterial culture) an additional round of antibiotics was completed two months later, which also failed to clear 1428’s infection. Numerous rounds of antibiotic treatment may not be a common commercial practice, but was appropriate within our research herd where previously we have been able to clear experimental S. aureus infections with this specific treatment and where milk is not used for human consumption. Interestingly, susceptibility testing of SA1428 by the ISU VDL, revealed susceptibility to several antibiotics (Ampicillin, Ceftiofur, Cephalothin, Erythromycin, Oxacillin, Penicillin, Penicillin/Novobiocin, Pirlimycin, Sulfadimethoxine, and Tetracycline). Mid-lactation, cow 1428 was treated off-label with a cytokine-based, PEG-gCSF therapy (Imrestor/Pegbovigrastim, Elanco, IN, USA), which was administered in two subcutaneous doses of 2.7 mL of 15 mg PEG-gCSF 7 days apart. While on-label use is designed for periparturient administration, our group was interested if the PEG-gCSF targeted neutrophil expansion would have an effect on 1428’s chronic infection. After PEG-gCSF treatment, circulating blood neutrophils increased from 2× 109 cells per liter of blood to 54 × 109 cells per liter of blood at their peak, 2 days post the second PEG-gCSF injection. In her infected quarter, cow 1428’s SCC also increased, from 1.11 × 106 cells per milliliter of milk to 5.17 × 106 cells per milliliter of milk, peaking 4 days post the second PEG-gCSF injection. Despite the increased presence of circulating neutrophils and milk SCC, cow 1428 did not clear the S. aureus infection. After being bred and confirmed pregnant, cow 1428 was dried off approximately 60 days prior to calving. In accordance with general dry-off practice, she was treated with cephapirin benzathine (ToMORROW, Boehringer Ingelheim, MO, USA). Seven days prior to cow 1428’s calving date she was treated again with an injection of PEG-gCSF, and again on the day of calving, as directed by on-label use of the product. Despite this treatment, 1428’s S. aureus infection presented immediately with the start of her second lactation.
We sought to characterize the infected mammary gland environment to identify phenotypes associated with SA1428 infection as compared to experimentally infected S. aureus. For the experimentally infected samples, we utilized resident Holsteins five weeks post experimental infection with S. aureus Newbould 305. For another ongoing study on the USDA National Animal Disease Center campus, eight Holstein cows were challenged by intramammary infusion in a single quarter with 150 CFU of S. aureus (Newbould). Subclinical, chronic infections developed in all cows. Five weeks after challenge, the Newbould infected cow with consistently high SCC values, and the Newbould infected cow with consistently low SCC values had milk samples collected for comparison along with milk from 1428’s naturally occurring infection. Over three consecutive days, 1428’s SCC for her infected quarter averaged 3.02 ± 0.78 × 106 cells per milliliter of milk, the high SCC cow averaged 12.59 ± 6.55 × 106 cells per milliliter of milk, and the low SCC cow averaged 0.24 ± 0.08 × 106 cells per milliliter of milk.
From S. aureus infected quarters 100–150 mL of milk was collected into 50 mL conical tubes. Samples were spun for 40 min, at 10,000 x g, at 4 °C to separate for pelleted milk cells for flow analysis and milk fat. Top milk fat layers were scraped into separate tubes, washed with PBS and protease inhibitor, and frozen for subsequent NET analysis.
Milk from the centrifuged samples was poured off, and cell pellets were placed on ice and resuspended in 1 mL media (L-glutamine, 10% FBS supplemented complete RPMI). Cell suspensions were layered over density gradients (Histopaque 1077, Sigma Aldrich, MO, USA, Cat No. 10771-500ML) spun for 20 min at 1500 x g, and had buffy coats removed leaving a highly neutrophil enriched cell pellet. Cell pellets were washed once with PBS and live cell counts were determined by cell counter (TC20 automated cell counter, BioRad, CA, USA). We used flow cytometry to evaluate the surface expression of MPO and L-selectin on neutrophils sourced from milk from infected quarters. To compare MPO and L-selectin expression over a range of SCC, we sampled milk from the Newbould infected cows with the highest and lowest SCC to compare with milk from cow 1428. Live milk cells were washed and resuspended in flow buffer (BioLegend, CA, USA, Cat. No. 420201). Individual primary, secondary, and directly conjugated antibodies were added to cell suspensions and incubated at room temperature for 15 min in the dark, with a flow buffer wash step between each antibody set. Samples were run on a Becton Dickinson LSR II flow cytometer and all analyses were performed with FlowJo software (FlowJo LLC, Ashland, OR, USA). Neutrophil gating was determined by forward and side scatter. Live, singlet milk cells were gated for CD45 (Monoclonal Antibody Center, Washington State University, USA. Cat. No. BOV2039). CD45+ cells were separately assessed for MPO (BioRad, Hercules, CA, USA Cat. No. VPA00193) and CD62L (BioLegend, San Diego, CA, USA Cat. No. 304824) surface expression. Flow cytometry of milk derived neutrophils from the three cows showed that cow 1428 had the greatest MPO surface expression (Fig. 1a). Between the Newbould infected cows, the high SCC cow also showed higher surface MPO expression compared to the low SCC cow (Fig. 1a). Surface expression of L-selectin revealed comparable levels on 1428 and the high SCC Newbould infected cow, but both were reduced compared to the low SCC Newbould infected cow (Fig. 1b).
Neutrophils are known to produce NETs with activation which results in cell death. To capture information about NET-forming neutrophils, we stained milk fat for DNA as described previously [16], which is indicative of NET formation from all three cows. Microscopy showed that 1428 had the greatest NET presence (Fig. 1c), compared to both Newbould infected cows (Fig. 1d, e). DNAse treated and healthy milk fat controls are shown in Fig. 1f, g. Samples were analyzed via confocal microscopy imaging using a Nikon A1R+ laser scanning microscope and NIS-Elements imaging software. Slide images are shown at the 20X objective, 75 numerical aperture, as imaged using a GaASP detector, 561 laser.
Cow 1428 was euthanized approximately 20 months after the first identification of infection by lethal injection of barbiturates by our institutional veterinarian. Gross pathology of the infected quarter of the mammary gland can be seen in Fig. 2a, b. The infected quarter was systematically sampled by obtaining samples from 12 different sites; 3 each from the proximal and distal gland body of the gland, 3 from the collecting duct region and 1 each from the gland cistern, teat cistern and streak canal as illustrated in Fig. 2c. Gross examination revealed multifocal abscesses and increased amounts of fibrous connective tissue, most notably in the collecting duct and gland cistern regions. Tissue samples (≤0.5 cm thick) were fixed by immersion in 10% neutral buffered formalin for 24 h, then transferred to 70% alcohol followed by standard paraffin embedding techniques. Paraffin embedded samples were cut to 4 μm thick sections, transferred to Superfrost Plus™ charged microscope slides (Thermo Fisher, MA, USA) and stained with hematoxylin and eosin (H&E). Adjacent sections were stained by the Hucker-Twort technique for visualization of Gram-positive and Gram-negative bacteria. Microscopically, samples from the teat sphincter and teat cistern were normal, with minimal if any inflammation (Fig. 2c) and no bacteria present, confirmed by Gram stain. Samples from the gland cistern and collecting duct regions contained multifocal suppurative to pyogranulomatous infiltrates. Some regions contained distinct infiltrates of only neutrophils surrounding colonies of Gram-positive cocci embedded in a brightly eosinophilic, homogenous matrix, which radiated outward; interpreted to be Splendore-Hoeppli reaction (Fig. 3a, b). In these same regions, there were also pyogranulomatous to granulomatous infiltrates arranged in nodules separated by prominent bands of fibrous connective tissue (Fig. 3c). These nodular infiltrates contained variable numbers of extracellular Gram-positive cocci. In the alveolar duct and body regions of the gland, numerous acini contained infiltrates of large numbers of neutrophils (Fig. 3d). In such acini, Gram-positive cocci were found both individually and in small colonies (Fig. 3e). Some glands were absent of inflammatory infiltrates, but one to several Gram-positive cocci could still be found adhered to or within epithelial cells (Fig. 3f).
To further document S. aureus strain SA1428, its genome was sequenced. The genome sequencing data have been deposited in NCBI Sequence Read Archive under accession number PRJNA609126. The de novo genome assembly is available at NCBI with the accession number CP048431-CP048432.
