Immuno‐modulating and anti‐viral properties of Tulathromycin in porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive‐strand RNA virus that grows in primary alveolar macrophages and causes acute pneumonia in pigs. PRRSV is a major concern in the swine industry with a total cost of productivity losses estimated at $600 million annually in the U.S.A alone. However, due to its high antigenic variability, and poorly understood immunopathogenesis, there is currently no treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections, including Actinobacillus pleuropneumoniae , begs the question of the value of antibiotics for the treatment of the disease it causes. Tulathromycin, a macrolide used for the treatment and prevention of respiratory disease in pigs and cattle, has been shown to exhibit potent immuno‐modulating properties [1]. We hypothesize that these may in turn attenuate the detrimental effects of PRRSV in porcine macrophages. Such findings will help characterize novel mechanism through which an anti‐microbial agent may deliver clinical benefits in the context of a viral infection. Aims 1) to determine if Tulathromycin has direct anti‐viral effects in PRRSV‐infected porcine macrophages. 2) to identify new immuno‐modulating effects of Tulathromycin in macrophages infected with PRRSV. Methods Porcine monocytes were isolated from peripheral blood drawn from the jugular vein of healthy piglets. Seven day‐old monocyte‐derived macrophages were treated with Tulathromycin (at a physiological concentration of 1 mg/ml [1]) or untreated (vehicle control) and incubated for 24 h at 37°C and 5% CO2. Macrophages were then cultured in media alone, or with PRRSV (multiplicity of infection, MOI 100:1) for 1 h. (1) Viral titers in supernatants, or intracellular titers inside macrophages, were measured at 1 h, 2 h, 24 h and 48 h post infection. Serial dilutions of 48h supernatants were performed (ranging from 10 −2 to 10 −7 pfu) and added to MARC‐145 cells at 100% confluence. (2) Macrophage activation was assessed by phenotypic observations under microscopy. (3) Macrophage phagocytic activity was measured using zymosan particles (1 μg/mL) for 24 hours. (4) Effects of Tulathromycin on viral induced necrosis was analyzed using lactate dehydrogenase (LDH) assay. Results (1) Tulathromycin did not change PRRSV particle forming unit (pfu) in macrophages at any time of infection ( 2.1×10 4 PFU/mL with tulathromycin versus 1.9×10 4 PFU/mL for control) (2). Exposure to PRRSV increases macrophage differentiation by more than four times compared to control. Pre‐treatment with tulathromycin significantly attenuated PRRSV‐induced macrophage activation (i.e. 1.85 times compared to untreated macrophages). (3) Macrophage phagocytic activity for zymosan was 25% lower in PRRSV‐infected macrophages versus uninfected control. Tulathromycin restored phagocytic impairment in PRRSV‐infected macrophages. (4) Tulathromycin reduced viral induced necrosis. Conclusion Our results demonstrate that Tulathromycin attenuates macrophage differentiation induced by PRRSV, restores PRRSV‐induced phagocytic impairment, and inhibits cell necrosis activated by the virus. These effects occur in the absence of a direct anti‐viral activity. Future research will assess whether and how these effects may alter the local production of pro‐inflammatory mediators in the lung, potentially conferring clinical benefits to Tulathromycin in the context of PRRSV‐induced pneumonia. Support or Funding Information Acknowledgements: Margaret Gunn endowment for Animal Health Research (U. of Calgary)

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