Safety and efficacy of a mesenchymal stem cell intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis

Abstract

Although, antibiotics are effective in the treatment of bovine mastitis, they do not address the regeneration of mammary glandular tissue and have been associated to the increment in antimicrobial resistance worldwide. Considering the necessity of alternative therapies for this disease of high economic impact and the reported regenerative and antibacterial effects of mesenchymal stem cell (MSCs), we evaluated the safety and efficacy of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis. In a safety trial, heifers were inoculated intramammarily with a 2.5 × 10⁷-suspension of bovine fetal AT-MSCs on experimental days 1 and 10. Animals were evaluated clinically on a daily basis during a 20-day experimental period and blood samples were collected for hemogram determination and peripheral blood leukocytes (PBLs) isolation. In an efficacy trial, Holstein Friesian cows were inoculated with S. aureus and treated intramammarily with vehicle (NEG; days 4 and 10), antibiotics (ATB; days 4 and 5) or a suspension of 2.5 × 10⁷ AT-MSCs (MSC; days 4 and 5). Cows were clinically evaluated daily and milk samples were collected for somatic cell count (SCC) and colony forming units (CFU). Blood samples were collected for serum haptoglobin and amyloid A determination. Intramammary administration of two doses of bovine fetal AT-MSCs in healthy cows did not induce changes in clinical or hematological variables, and gene expression profiles in PBLs associated to activation (CD4, CD8, CD25, CD62L and CD69) and proinflammatory cytokines (CCL2, CCL5, IL2, CXCL3, IFNγ, and TNFα). Quarters of MSC group of cows had similar SCC log/mL in milk compared to infected quarters of ATB or NEG cows. However, quarters of MSC cows had lower CFU log/mL in milk compared to quarters of NEG cows. Intramammarily inoculation of repeated doses of 2.5 × 10⁷ allogenic AT-MSCs did not induce clinical or immunological response in healthy cows. Moreover, MSC-intramammary treatment reduced bacterial count in milk of cows with S. aureus clinical mastitis compared to untreated cows. This work provides initial evidence for the safety and efficacy of an allogenic MSC-based intramammary therapy for the treatment of bovine mastitis.

Figure 1

Experimental designs for safety and efficacy studies of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis.

Figure 2

Gene expression profiles in peripheral blood leukocytes (PBLs) associated to activation and proinflammatory cytokines in healthy dairy heifers treated with two doses of allogenic bovine fetal AT-MSCs. No significant differences (P > 0.05) were found in gene expression profiles in PBLs associated to activation (A) and proinflammatory cytokines (B) in blood samples collected from healthy dairy heifers (n = 8) every five days during a 20-Day period. Black arrows represent AT-MSCs intramammary inoculation at days 1 and 10.

Figure 3

Somatic cell count (SCC) and colony forming units (CFU) in milk from mammary quarters with experimentally induced clinical mastitis by S. aureus and treated with bovine fetal AT-MSCs (MSC; n = 10 quarters), antibiotics (ATB; n = 10 quarters) or vehicle (NEG; n = 10 quarters). Non-infected mammary quarters (CON, n = 30 quarters) were used as negative controls. (A) Mean SCC log/mL from NEG, ATB and MSC infected mammary quarters were higher (Days 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, and 15; P < 0.05) compared to CON mammary quarters. (B) Mean CFU log/mL in infected mammary quarters of MSC and ATB treatments were lower (Days 6, 7, 8, 9, and 10; P < 0.05) compared to NEG mammary quarters. Different superscripts (*, a,b,c) indicate significant differences between treatments. NEG, infected mammary quarters treated with vehicle on experimental days 4 and 10 (black arrows); ATB, infected mammary quarters treated with 50 mg of pirlimycin on experimental days 4 and 5 (black arrows); MSC, infected mammary quarters treated with a suspension of 2.5 × 10⁷ of AT-MSCs on experimental days 4 and 5 (black arrows); CON, non-infected mammary quarters inoculated with 1 mL of PBS on day 1 as a control.