Colloidal Iron Oxide Formulation for Equine Hoof Disinfection

Abstract

The presence of bacteria of various origins on horse hoofs enables the onset of infections following trauma or even post-surgical wounds. Thus, the analysis of new antibacterial substances is of fundamental importance. In this study, the antibacterial efficacy of Iron Animals (IA), a stable colloidal suspension of iron oxide, organic acids, and detergents, was tested in vitro and in vivo. In vitro assays were performed to test the unspecific inhibitory effect of IA on both gram-positive and gram-negative bacteria monitoring the microorganism growth by spectrophotometry (optical density OD600) at 37 °C for 24 h. In vivo test consists on the quantification of the bacterial load in colony forming units per gram (CFU/g) of specimens collected from the frog region of the anterior hooves of 11 horses. Sampling followed the application of four disinfectant protocols consisting of two consecutive 3 min scrubs with 50 mL of 10% Povidone-iodine (PI) or 4% Chlorhexidine (CHx), with or without an additional application for 15 min of 10 mL of Iron Animals (PI+IA and CHx+IA). In vitro, IA completely suppressed the bacterial growth of all the tested microorganisms, resulting in effectiveness also against CHx-resistant bacteria, such as Staphylococcus aureus. In vivo, PI emerged as an ineffective protocol; CHx was effective in 18% of cases, but with the addition of IA (CHx + IA) its use emerged as the best disinfectant protocol for horse hoof, achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improves the effectiveness of both disinfectants leading to the highest bactericidal activity in 82% of cases.

Keywords: chlorhexidine; hoof; horse; povidone-iodine; pre-surgical disinfection.

Figure 1

Bacterial growth of Staphylococcus aureus ATCC 29,213 (—), S. aureus 393 (—), Pseudomonas aeruginosa L2 (—), P. aeruginosa L4 (—) in the absence (A) and presence (B) of Iron Animals. ABS (arbitrary units) = optical density OD₆₀₀ − blank OD₆₀₀.

Figure 2

Microbiological analysis of the fragments from the front and rear hoof sole of one horse. The plates show the microbial growth on Nutrient Agar, after incubation at 37 °C for 24 h. (A) Sample from the left front hoof sole after superficial cleaning; (B) Sample from the right front hoof sole after two consecutive 3 min scrubs with Povidone-iodine (PI); (C) I, Sample from the left rear hoof sole after superficial cleaning; II, Sample from the right rear hoof sole after application of 10 mL Iron Animals (IA) for 15 min.

Figure 3

Representation of the average residual bacterial load after different protocols. Data were normalized for the residual bacterial load achieved in Povidone-iodine (PI) protocol, which was used as a reference procedure and referred to as 100%. PI: two consecutive 3 min scrubs with PI; Chlorhexidine (CHx): two consecutive 3 min scrubs with CHx; PI + Iron Animals (IA) and CHx + IA represent PI and CHx protocol followed by the application of 10 mL of IA for 15 min.

Figure 4

Graphical representation of the fraction of cases in which each protocol obtained the lowest bacterial load within the hooves of the same animal. PI: two consecutive 3 min scrubs with Povidone-iodine (PI); Chlorhexidine (CHx): two consecutive 3 min scrubs with CHx; PI + Iron Animals (IA) and CHx + IA represent PI and CHx protocol followed by the application of 10 mL of IA for 15 min.