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      Evaluating the effectiveness of commercial teat disinfectant products sold in Ireland using the disc diffusion method

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            Abstract

            Evaluation of teat disinfectant products for their effectiveness against the most prevalent mastitis-causing bacteria is important to identify the most effective ingredients against specific bacterial strains. Ninety-six commercially available teat disinfectant products were tested against three bacterial strains associated with mastitis in Ireland (Staphylococcus aureus, Streptococcus uberis and Escherichia coli) using the disc diffusion method. Products were reclassified by active ingredients (n = 9) for analysis. These ingredient groups included: chlorhexidine (n = 25), chlorine dioxide (n = 5), diamine (n = 1), iodine (n = 13), iodine combined with lactic acid (n = 5), lactic acid (n = 15), lactic acid combined with chlorhexidine (n = 21), lactic acid combined with hydrogen peroxide (n = 1) and lactic acid combined with salicylic acid (n = 10). The ingredient group chlorine dioxide resulted in the greatest zones of inhibition for all three bacterial strains. An individual product containing a combination of lactic acid and hydrogen peroxide resulted in the greatest zone of inhibition for Sta. aureus and Str. uberis, whereas a specific product within the chlorine dioxide group resulted in the greatest zones of inhibition for E. coli. High concentrations of active ingredient did not necessarily increase the effectiveness for the majority of teat disinfectant products. It is possible to use the disc diffusion method to evaluate/screen a large number of teat disinfectant products prior to conducting field trials to establish the products’ ability to reduce intramammary infections (IMI).

            Main article text

            Introduction

            Bovine mastitis or intramammary infection (IMI) is an inflammatory reaction which is caused by different microbial pathogens which can gain entry into the mammary gland through the teat canal (Berry & Meaney, 2006; Keane et al., 2013). However, despite decades of advances regarding the prevention and treatment of mastitis, it continues to be one of the main causes of impaired milk quality, decreased production, reduced profit and animal morbidity and mortality (Ruegg, 2012). There is a large variation in the pathogens identified within countries, which are associated with mastitis, and these differences may be due to different veterinary and laboratory services and farmer management practises (Zadoks & Fitzpatrick, 2009). In most countries, the most common bacteria associated with mastitis are Str. agalactiae, Str. dysgalactiae, Str. uberis, Str. aureus and Escherichia coli (Zadoks & Fitzpatrick, 2009). A study by Keane et al. (2013) identified Sta. aureus, Str. uberis and E. coli as the main bacteria associated with clinical mastitis on Irish dairy farms. This study also found that Str. uberis and E. coli were more commonly associated with clinical mastitis than Sta. aureus.

            Implementation of an effective mastitis control plan can help to prevent and reduce incidence of mastitis and reduce horizontal transmission of bacteria from cow to cow and within the environment. These control measures include hygienic milking and housing conditions, routine milking machine maintenance, teat disinfection pre- and post-milking, dry cow therapy, isolation of infected animals and cow culling (Hillerton & Booth, 2018). Studies have reported that the use of pre-milking teat cleaning regimes, using teat disinfectants, can reduce the bacterial load on the teat skin surface (Gibson et al., 2008; Mišeikienė et al., 2015; Baumberger et al., 2016). Previous studies also showed that pre-milking teat disinfection helped to reduce mastitis caused by environmental bacteria (Pankey, 1989; Oliver et al., 1993a,b, 2001) and mastitis caused by Streptococcus spp. and Gram-negative bacteria (Oliver et al., 1993b). These studies were undertaken when cows were indoors; however, where cows were grazed on pastures, little to no improvement on mastitis incidence levels was observed (Williamson & Lacy-Hulbert, 2013; Gleeson et al., 2018). Post-milking teat disinfection is important for the control of contagious mastitis in herds (Breen, 2019). Contagious bacteria tend to be spread from cow to cow during milking via the milking machine and by the milker’s hands. A study by Williamson and Lacy-Hulbert (2013) demonstrated that cows receiving post-milking disinfection had a lower rate of IMIs, caused by Sta. aureus, Str. uberis, Corynebacterium spp. and coagulase-negative staphylococci, than cows that did not receive post-milking disinfection. The success of teat disinfection in reducing new IMIs may also be influenced by the product active ingredient.

            At present, the main knowledge regarding teat disinfectants relates to iodine as it is a broad-spectrum disinfectant and has been proven to be effective against mastitis and new IMIs (Oliver et al., 1991; Boddie et al., 2004; Böhm et al., 2017). However, alternative ingredients to iodine are now desirable due to concerns regarding iodine residues in milk which may be destined for infant milk formula manufacturing. Unfortunately, little knowledge is known regarding the effectiveness of these ingredients and products within an Irish context. There are many test methods available to measure the effectiveness of teat disinfection products. For regulatory purposes, teat disinfection products are required to be evaluated by the BS EN 1656 laboratory test method, known as a European Standards test. The National Mastitis Council (NMC) recommends the use of the experimental challenge and natural exposure protocols as they are useful for demonstrating field efficacy in reducing new IMI and mastitis. However, these tests may be limited to showing efficacy against specific organisms present on individual farms (Lopez-Benavides et al., 2012). The disc diffusion laboratory method can be used to screen disinfection products against a broad range of mastitis pathogens (Garvey et al., 2017; Fitzpatrick et al., 2019a). Screening/testing products using such a method can identify effective products before time-consuming and expensive field tests are undertaken. The objective of this study was to independently screen the effectiveness of 96 commercially available teat disinfection products, against the three main bacteria associated with mastitis in Ireland, Sta. aureus, Str. uberis and E. coli, using the disc diffusion method.

            Materials and methods

            Teat disinfectant information

            Ninety-six commercially available teat disinfectant products (Table 1), with different active ingredients of varying concentrations, were tested against mastitis-causing bacteria, using the disc diffusion method. The teat disinfectant products were either ready-to-use (RTU) (n = 82), concentrate (conc.) products (n = 9) or required activation before use (n = 5). Concentrate products were diluted using sterile distilled water according to the manufacturer’s recommendation to avoid possible issues with water hardness or contaminated water. Five chlorine dioxide-based products (products 11, 70, 89, 90 and 95) were mixed with an activator before use according to the manufacturer’s recommendations. The disinfectant products used were recommended either for both pre-/post-milking teat disinfection (n = 49), pre-milking teat disinfection only (n = 3) or post-milking disinfection only (n = 44). Concentrations of active ingredients are declared where indicated by the manufacturer on the product label.

            Table 1:

            Test teat disinfectant product code and active ingredient, as declared by the manufacturer on product label

            Product#Ingredient (w/w)Pre or post
            Arkshield1 75% Lactic acid/0.3% chlorhexidinePre/post
            Arrabawn Udder Guard1 400.5% ChlorhexidinePre/post
            Bacto-Lac1 315% Lactic acid/0.05% chlorhexidinePre/post
            Barri-max1 652.4% Lactic acidPost
            Biolac Pre-Post1 440.25% Lactic acid/0.03% salicylic acidPre/post
            Biolac Pre-Post1 590.25% Lactic acid/0.03% salicylic acidPre/post
            Bisept2 700.05% Chlorine dioxidePre/post
            Blue Barrier Spray1 49Lactic acid/0.6% chlorhexidine3 Post
            Blu-gard N Spray1 153.46% Lactic acidPost
            C-Dip1 610.53% ChlorhexidinePost
            Co-op Source Duo-Teat Shield1 392% Lactic acid/0.3% chlorhexidinePre/post
            D 4 Iodine4 190.5% IodinePost
            Dairy Pro UltraDip1 743% Lactic acidPost
            DairyLac SA1 763% Lactic acidPost
            Deosan Mastocide1 320.5% ChlorhexidinePost
            Deosan Summer Teat Care1 330.425% ChlorhexidinePost
            Deosan Super Iodip4 340.5% IodinePost
            Deosan Teat Foam Advance1 130.6% ChlorhexidinePre/post
            Deosan Teatcare Plus1 140.425% ChlorhexidinePost
            Deosan Triathalon1 811.76% Lactic acidPre/post
            Dermalac Emprasan1 270.25% Lactic acid/salicylic acid3 Pre/post
            Dual Dip Supreme1 47Lactic acid/0.6% chlorhexidine3 Pre/post
            Dual Dip1 452% Lactic acid/0.3% chlorhexidinePre/post
            Duo-cel1 382.5% Lactic acid/0.3% chlorhexidinePre/post
            Duogold1 172% Lactic acid/0.3% chlorhexidinePre/post
            Duo-Teat Shield1 252% Lactic acid/0.3% chlorhexidinePre/post
            Emprasan dual1 530.25% Lactic acid/salicylic acid3 Pre/post
            Flexigard Spray1 944% Lactic acidPost
            Fortress Protect Film1 733% Lactic acid/0.2% chlorhexidinePost
            Gold Glycodip XL1 620.5% Iodine/1% lactic acidPost
            Hamra Red1 120.42% ChlorhexidinePost
            Hexa-cel RTU1 420.52% ChlorhexidinePre/post
            Hexaguard1 10.74% ChlorhexidinePre/post
            Hexaklene R1 20.5% ChlorhexidinePre/post
            Hexa-Spray1 820.5% ChlorhexidinePre/post
            Hypraspray1 872% Lactic acid/0.03% chlorhexidinePre/post
            Hypred Quick Spray1 202% Lactic acid/0.1% salicylic acidPre/post
            Ioguard4 30.5% IodinePost
            Ioklar Multi1 920.25% IodinePre/post
            Io-Shield D1 911.35% IodinePost
            Io-Shield Spray1 930.5% IodinePost
            Kenocidin Spray and Dip1 90.5% ChlorhexidinePost
            Kenolac SD1 803.6% Lactic acidPost
            Kenolac1 103.6% Lactic acidPost
            Kenomint SD1 780.5% ChlorhexidinePost
            Kenomint1 770.5% ChlorhexidinePost
            Kenomix SD2 890.0157% Chlorine dioxidePost
            Kenomix2 110.0157% Chlorine dioxidePost
            Kenopure1 793.2% Lactic acidPre
            Lactic Lather1 461.6% Lactic acid/hydrogen peroxide3 Pre
            Lacto dual1 362.5% Lactic acid/1.5% chlorhexidinePre/post
            Lacto-cel1 352.4% Lactic acidPre/post
            Lacto-Mil1 965% Lactic acidPre/post
            Lactospray1 42.4% Lactic acidPre/post
            Lanodip 4 XL1 300.5% Iodine/0.5% lactic acidPost
            Lanodip Pre-Post1 550.29% Iodine/0.8% lactic acidPre/post
            Lely Quaress-Cura1 433% Lactic acid/salicylic acid3 Post
            Luxdip 50B1 690.5% IodinePost
            Masocare Platinum1 850.54% IodinePre/post
            Masodine Concentrate4 830.5% IodinePre/post
            Masodip Platinum1 840.436% ChlorhexidinePre/post
            Maxadine C4 80.5% IodinePost
            Maxidine RTU1 370.5% IodinePost
            Nano Dual1 281.93% Lactic acid/0.2% chlorhexidinePre/post
            Novo Dual1 294% Lactic acid/0.27% chlorhexidinePre/post
            Novodip1 604.9% Lactic acid/1.28% chlorhexidinePost
            Novospray1 544.9% Lactic acid/0.3% chlorhexidinePost
            Prefoam+1 212% Lactic acid/0.1% salicylic acidPre
            Protect Pre Post1 753% Lactic acid/0.25% chlorhexidinePre/post
            PureChem Chlorhexidine Summer Grade1 521.49% ChlorhexidinePre/post
            PureChem Chlorhexidine1 481.49% ChlorhexidinePost
            PureChem Dual Dip1 511% Lactic acid/1.49% chlorhexidinePre/post
            PureChem Iodophor4 500.5% IodinePost
            Quatro1 410.5% ChlorhexidinePre/post
            SalvoDip B1 712.4% Lactic acidPost
            Salvohex1 672% Lactic acid/0.3% chlorhexidinePost
            Salvospray1 682.4% Lactic acidPre/post
            SensoDip 501 160.5% ChlorhexidinePost
            SensoDip1 720.5% ChlorhexidinePre/post
            Sensospray1 660.5% ChlorhexidinePost
            Silkdip4 240.5% IodinePost
            Summer C-Dip1 580.5% ChlorhexidinePost
            Super Cow Teat Foam1 260.6% DiaminePre/post
            Supergold1 180.5% ChlorhexidinePre/post
            Supreme1 642.5% Lactic acid/0.37% chlorhexidinePre/post
            Sure Spray Duo1 62% Lactic acid/0.3% chlorhexidinePre/post
            Surespray1 50.5% ChlorhexidinePre/post
            Synofilm1 888% Lactic acidPost
            Teat Gard C1 630.5% ChlorhexidinePre/post
            TriCide Gold1 570.15% Iodine/1% lactic acidPre/post
            TriCide1 560.15% Iodine/1% lactic acidPost
            Uddergold2 900.32% Acidified sodium chloritePost
            Valiant2 950.038% Sodium chloridePost
            Virolac Film1 232% Lactic acid/0.1% salicylic acidPost
            Virolac Spray1 862% Lactic acid/0.1% salicylic acidPre/post
            Virolac Concentrate4 222% Lactic acid/0.1% salicylic acidPre/post

            1Ready to use (RTU).

            2Requires activation before use.

            3The concentration of some active ingredients for combination products was not declared by the manufacturer.

            4Concentrate.

            # = product number.

            Bacterial strain identification

            The bacteria applied in this study were isolated from the teat skin of cows within the Teagasc Moorepark research herd, by taking skin swab samples from lactating cows’ teats using moistened cotton swabs, according to NMC (NMC, 2017) guidelines. The isolates were gram stained and bacterial identification was carried out using biochemical tests including lactose fermentation, motility test medium (Sigma-Aldrich, Dublin, Ireland), catalase and oxidase tests, tube coagulase (Sigma-Aldrich, Dublin, Ireland) and growth/reaction on various types of agars including blood agar (Sigma-Aldrich, Dublin, Ireland), MacConkey (Merck Millipore, Cork, Ireland), Baird Parker (Merck KGaA64271, Darmstadt, Germany) and modified Edwards agar (Oxoid 3M0027, Hampshire, UK), Simmons citrate agar (Sigma-Aldrich, Dublin, Ireland), CAMP esculin agar (Sigma-Aldrich, Dublin, Ireland) and triple sugar iron agar (Sigma-Aldrich, Dublin, Ireland). Analytical Profile Index-Staph (API-Staph Kit, bioMerieux, Marcy-l’Etoile, France) and API 20 tests (API, bioMerieux, Marcy-l’Etoile, France) were also used according to the manufacturer’s instructions.

            Disc diffusion method

            The disc diffusion method was used to determine the ability of the teat disinfectant products to inhibit bacterial growth. This method was chosen based on a previous study that demonstrated that the method can effectively screen/evaluate a number of teat disinfectant products in a short period of time (Fitzpatrick et al., 2019a).

            The disc diffusion method was performed using the techniques as described by Fitzpatrick et al. (2019a). Each bacterial strain isolated was grown on Mueller Hinton (MH) agar (Sigma-Aldrich, Ireland) plates with blank filter paper discs (three per plate; Cruinn, Dublin, Ireland) impregnated with a different teat disinfectant product. The experiment was independently repeated over 3 d, with three plate replications for each product against each bacterial strain tested. A pilot study was performed to determine the inclusion of sterile skimmed milk or sterile bovine serum albumin (BSA) as an interfering substance to imitate the environment that the teat disinfectants would be used in. In this pilot study, the disc diffusion method was modified to allow for the addition of the aforementioned interfering substances. This modification involved adding the adjusted bacterial suspension to the interfering substance/organic matter suspension. The method was then carried out as described above. The zone of no growth (zone of inhibition) (measured in millimetres [mm], using a digital calliper [RS digital calliper 600/880, Mitutoyo Digimatic, Hampshire, UK]) around the disc is a measure of the ability of the teat disinfectant product to inhibit the growth of the test bacterial strain.

            Statistical analysis

            Statistical analysis was carried out using SAS for Windows, version 9.4 (SAS Institute Inc., Cary, NC, USA; SAS, 2014). The results were analysed using the PROC GLIMMIX procedure. Pair-wise comparisons were adjusted for multiplicity effect using simulation procedures to adjust P-values. Residual checks were made to ensure assumptions of analysis were met. This was used to determine the difference in susceptibility or resistance of bacterial species and differences in zones of inhibition between teat disinfectant products within ingredient groups. Products used within the study were reclassified by active ingredients (n = 9) to minimise/control the occurrence of type II errors during analysis. Comparisons between ingredient groups and between products within each ingredient group were compared using LSMEANS in the PROC GLIMMIX procedure. These ingredient groups included: chlorhexidine (n = 25), chlorine dioxide (n = 5), diamine (n = 1), iodine (n = 13), iodine combined with lactic acid (n = 5), lactic acid (n = 15), lactic acid combined with chlorhexidine (n = 21), lactic acid combined with hydrogen peroxide (n = 1) and lactic acid combined with salicylic acid (n = 10).

            Results

            The bacteria isolated from teat skin swab samples and identified in this study were found to be Sta. aureus, Str. uberis and E. coli. Furthermore, the pilot study showed that the use of either of the two different interfering substances did not have a negative impact on the effectiveness of the teat disinfectants used. Therefore, no interfering substance was included in the current study.

            The average zone of inhibition for each product group can be seen in Figure 1. Lactic acid combined with hydrogen peroxide achieved the largest zones of inhibition for all three bacterial strains (Str. uberis [27.9 mm], Sta. aureus [25.1 mm] and E. coli [19.3 mm]). This was followed by the ingredient group chlorine dioxide (Str. uberis [21.3 mm], Sta. aureus [20.0 mm] and E. coli [18.1 mm]). Chlorhexidine group and diamine resulted in the smallest bacterial inhibitions for Str. uberis (17.9 mm and 16.1 mm, respectively), with a combination of lactic acid and salicylic acid achieving significantly smaller zones of inhibition against Sta. aureus (13.2 mm) compared to chlorine dioxide (P < 0.05). For E. coli, the ingredient iodine and lactic acid resulted in a smaller level of bacterial inhibition (10.9 mm) compared to a combination of lactic acid and hydrogen peroxide and chlorine dioxide (P < 0.05).

            Figure 1.

            Least squares means of the zones of inhibition (mm) of (A) Str. uberis, (B) Sta. aureus and (C) E. coli across all teat disinfectant active ingredient groups tested by the disc diffusion method. Error bars indicate SEM. a,b,c,d,e,f Inhibitions not sharing the same superscript for each bacterial strain were significantly different (P < 0.05).

            Within the study, two ingredient groups (diamine and lactic acid combined with hydrogen peroxide) contained only one product each. The ingredient group, diamine, demonstrated a smaller level of bacterial inhibition, with an overall average of 16.1 mm, 14.5 mm and 13.6 mm for Str. uberis, Sta. aureus and E. coli, respectively. The ingredient group which included a combination of lactic acid and hydrogen peroxide achieved a large bacterial inhibition against Str. uberis (27.9 mm), Sta. aureus (25.1 mm) and E. coli (19.3 mm). Bacterial inhibition of teat disinfectant products against different bacterial strains within each ingredient group can be observed in Table 2.

            Table 2:

            Least squares means of the zones of inhibition (mm) for 96 teat disinfectant products, categorised by ingredient group, against Str. uberis, Sta. aureus and E. coli

            Product#Ingredient (w/w) Str. uberis Sta. aureus E. coli
            Chlorhexidine products

            Arrabawn Udder Guard1 400.5% Chlorhexidine16.6 d,e,f,g 15.3 b,c,d 16.2 b,c,d
            C-Dip1 610.53% Chlorhexidine17.6 d,e,f,g 15.7 b,c,d 15.6 b,c,d,e
            Deosan Mastocide1 320.5% Chlorhexidine19.1 a,b,c,d,e,f 17.4 a,b,c 16.7 b,c,d
            Deosan Summer Teat Care1 330.425% Chlorhexidine17.4 d,e,f,g 16.7 a,b,c,d 17.9 a,b,c,d
            Deosan Teat Foam Advance1 130.6% Chlorhexidine19.5 a,b,c,d,e 16.1 a,b,c,d 16.3 b,c,d
            Deosan Teatcare Plus1 140.425% Chlorhexidine15.8 g 16.9 a,b,c,d 17.6 a,b,c,d
            Hamra Red1 120.42% Chlorhexidine17.8 b,c,d,e,f,g 16.0 b,c,d 15.9 b,c,d,e
            Hexa-cel RTU1 420.52% Chlorhexidine15.9 f,g 16.1 a,b,c,d 15.1 b,c,d,e
            Hexaguard1 10.74% Chlorhexidine21.4 a 17.7 a,b 18.3 a,b,c
            Hexaklene R1 20.5% Chlorhexidine20.9 a,b,c 15.9 b,c,d 20.6 a
            Hexa-Spray1 820.5% Chlorhexidine17.3 d,e,f,g 14.0 d 12.3 e
            Kenocidin Spray/Dip1 90.5% Chlorhexidine17.0 d,e,f,g 16.2 a,b,c,d 14.2 d,e
            Kenomint1 770.5% Chlorhexidine17.7 c,d,e,f,g 16.0 b,c,d 16.3 b,c,d
            Kenomint SD1 780.5% Chlorhexidine19.1 a,b,c,d,e,f 14.6 c,d 14.5 c,d,e
            Masodip Platinum1 840.436% Chlorhexidine16.4 e,f,g 17.9 a,b 14.4 d,e
            PureChem Chlorhexidine1 481.49% Chlorhexidine15.6 g 15.0 b,c,d 14.8 b,c,d,e
            PureChem Chlorhexidine Summer Grade1 521.49% Chlorhexidine17.4 d,e,f,g 16.3 a,b,c,d 16.3 b,c,d
            Quatro1 410.5% Chlorhexidine16.1 f,g 16.6 a,b,c,d 16.5 b,c,d
            SensoDip1 720.5% Chlorhexidine17.0 d,e,f,g 15.9 b,c,d 15.9 b,c,d,e
            SensoDip 501 160.5% Chlorhexidine18.4 a,b,c,d,e,f,g 17.4 a,b,c 17.3 a,b,c,d
            Sensospray1 660.5% Chlorhexidine16.9 d,e,f,g 17.1 a,b,c,d 14.9 b,c,d,e
            Summer C-Dip1 580.5% Chlorhexidine19.4 a,b,c,d,e, 15.1 b,c,d 14.5 c,d,e
            Supergold1 180.5% Chlorhexidine21.0 a,b 16.5 a,b,c,d 18.5 a,b
            Surespray1 50.5% Chlorhexidine19.8 a,b,c,d 19.2 a 18.2 a,b,c
            Teat Gard C1 630.5% Chlorhexidine16.0 f,g 15.2 b,c,d 15.4 b,c,d,e

            Chlorine dioxide products

            Bisept2 700.05% Chlorine dioxide19.4 c 18.5 b 12.2 c
            Kenomix2 110.0157% Chlorine dioxide22.6 a 18.2 b 19.3 a,b
            Kenomix SD2 890.0157% Chlorine dioxide21.0 b 21.4 a,b 20.5 a,b
            Uddergold2 900.32% Acidified sodium chlorite20.9 b,c 19.5 a,b 17.3 b
            Valiant2 950.038% Sodium chloride22.8 a 22.4 a 21.5 a

            Diamine products

            Super Cow Teat Foam1 260.6% Diamine16.114.513.6

            Iodine products

            D 4 Iodine3 190.5% Iodine21.4 a,b,c 16.1 a,b,c 10.9 b
            Deosan Super Iodip3 340.5% Iodine20.0 b,c,d,e 15.2 b,c,d,e 11.7 b
            Ioguard3 30.5% Iodine21.0 a,b,c,d 13.5 c,d,e 10.2 b,c
            Ioklar Multi1 920.25% Iodine12.1 g 9.2 f 7.5 c
            Io-Shield D1 911.35% Iodine19.0 d,e,f 18.2 a 16.3 a
            Io-Shield Spray1 930.5% Iodine17.3 f 14.5 b,c,d,e 12.8 b
            Luxdip 50B1 690.5% Iodine19.2 c,d,e,f 13.7 c,d,e 11.4 b
            Masocare Platinum1 850.54% Iodine19.3 c,d,e,f 14.5 b,c,d,e 12.4 b
            Masodine Concentrate3 830.5% Iodine20.7 a,b,c,d,e 15.5 b,c,d 10.8 b
            Maxadine C3 80.5% Iodine23.0 a 15.8 a,b,c,d 11.9 b
            Maxidine RTU1 370.5% Iodine18.9 d,e,f 13.3 d,e 11.4 b
            PureChem Iodophor3 500.5% Iodine18.6 e,f 12.6 f 10.5 b
            Silkdip3 240.5% Iodine21.6 a,b 17.1 a,b 12.0 b

            Iodine and lactic acid products

            Gold Glycodip XL1 620.5% Iodine/1% lactic acid21.6 a 15.3 a,b 12.0 a
            Lanodip 4 XL1 300.5% Iodine/0.5% lactic acid21.9 a 16.5 a 12.1 a
            Lanodip Pre-Post1 550.29% Iodine/0.8% lactic acid21.1 a 13.3 b,c 10.1 b
            TriCide1 560.15% Iodine/1% lactic acid21.1 a 13.0 c 10.0 b
            TriCide Gold1 570.15% Iodine/1% lactic acid20.3 a 13.2 c 10.1 b

            Lactic acid products

            Barri-max1 652.4% Lactic acid19.7 b,c,d 16.3 a,b,c 13.6 b
            Blu-gard N Spray1 153.46% Lactic acid19.9 b,c,d 15.6 b,c 11.4 b,c,d
            Dairy Pro UltraDip1 743% Lactic acid17.1 e,f 16.1 a,b,c 12.1 b,c,d
            DairyLac SA1 763% Lactic acid18.8 d,e,f 14.5 c 11.8 b,c,d
            Deosan Triathalon1 811.76% Lactic acid19.7 b,c,d 14.2 c 11.4 b,c,d
            Flexigard Spray1 944% Lactic acid22.4 a 19.2 a 18.2 a
            Kenolac1 103.6% Lactic acid22.3 a 19.3 a 10.0 d
            Kenolac SD1 803.6% Lactic acid19.a c,d,e 15.0 b,c 13.2 b,c
            Kenopure1 793.2% Lactic acid21.6 a,b 16.4 a,b,c 11.2 b,c,d
            Lacto-cel1 352.4% Lactic acid19.9 b,c,d 15.9 b,c 12.1 b,c,d
            Lacto-Mil1 965% Lactic acid19.1 c,d,e 13.7 c 10.6 c,d
            Lactospray1 42.4% Lactic acid21.3 a,b,c 19.2 a 10.2 d
            SalvoDip B1 712.4% Lactic acid19.6 b,c,d 16.6 a,b,c 11.5 b,c,d
            Salvospray1 682.4% Lactic acid18.8 d,e,f 17.8 a,b 11.5 b,c,d
            Synofilm1 888% Lactic acid16.6 f 19.3 a 17.3 a

            Lactic acid and chlorhexidine products

            Arkshield1 75% Lactic acid/0.3% chlorhexidine21.6 a,b,c 18.9 a,b,c 15.4 b,c,d
            Bacto-Lac1 315% Lactic acid/0.05% chlorhexidine18.8 c,d,e,f 15.9 d,e 14.1 c,d
            Blue Barrier Spray1 49Lactic acid/0.6% chlorhexidine4 22.3 a 21.7 a 20.3 a
            Co-op Source Duo-Teat Shield1 392% Lactic acid/0.3% chlorhexidine18.1 d,e,f 16.5 c,d,e 15.4 b,c,d
            Dual Dip1 452% Lactic acid/0.3% chlorhexidine18.2 d,e,f 16.0 d,e 15.0 b,c,d
            Dual Dip Supreme1 47Lactic acid/0.6% Chlorhexidine4 21.8 a,b 21.3 a,b 18.5 a,b
            Duo-cel1 382.5% Lactic acid/0.3% chlorhexidine18.5 d,e,f 16.4 c,d,e 16.2 b,c
            Duogold1 172% Lactic acid/0.3% chlorhexidine19.7 a,b,c,d,e 16.8 c,d,e 17.3 a,b,c
            Duo-Teat Shield1 252% Lactic acid/0.3% chlorhexidine17.9 d,e,f 16.9 c,d,e 16.8 a,b,c
            Fortress Protect Film1 733% Lactic acid/0.2% chlorhexidine20.3 a,b,c,d,e 17.6 c,d,e 13.9 c,d
            Hypraspray1 872% Lactic acid/0.03% chlorhexidine19.1 b,c,d,e 16.7 c,d,e 12.2 d
            Lacto dual1 362.5% Lactic acid/1.5% chlorhexidine18.2 d,e,f 15.2 e 16.6 b,c
            Nano Dual1 281.93% Lactic acid/0.2% chlorhexidine21.6 a,b,c 16.9 c,d,e 14.8 c,d
            Novo Dual1 294% Lactic acid/0.27% chlorhexidine20.8 a,b,c,d 17.5 c,d,e 15.6 b,c,d
            Novodip1 604.9% Lactic acid/1.28% chlorhexidine19.3 b,c,d,e 19.1 a,b,c 16.0 b,c
            Novospray1 544.9% Lactic acid/0.3% chlorhexidine20.0 a,b,c,d,e 18.4 b,c,d 16.3 b,c
            Protect Pre Post1 753% Lactic acid/0.25% chlorhexidine21.7 a,b 18.2 c,d 14.0 c,d
            PureChem Dual Dip1 511% Lactic acid/1.49% chlorhexidine16.2 f 16.3 c,d,e 14.5 a
            Salvohex1 672% Lactic acid/ 0.3% chlorhexidine17.8 e,f 16.2 c,d,e 16. 8 a,b,c
            Supreme1 642.5% Lactic acid/0.375% chlorhexidine19.2 b,c,d,e 16.8 c,d,e 15.4 b,c,d
            Sure Spray Duo1 62% Lactic acid/0.3% chlorhexidine18.6 d,e,f 17.2 c,d,e 15.4 b,c,d

            Lactic acid and hydrogen peroxide products

            Lactic Lather1 461.6% Lactic acid/ hydrogen peroxide4 27.925.119.3

            Lactic acid and salicylic acid products

            Biolac Pre-Post1 440.25% Lactic acid/0.03% salicylic acid19.7 a,b 16.0 a 11.4 b
            Biolac Pre-Post1 590.25% Lactic acid/0.03% salicylic acid21.0 a 15.1 a 11.8 b,c
            Dermalac Emprasan1 270.25% Lactic acid/salicylic acid4 21.1 a 14.3 a,b,c 11.0 b,c
            Emprasan dual1 530.25% Lactic acid/salicylic acid4 19.6 a,b 14.8 a,b 16.0 a
            Hypred Quick Spray1 202% Lactic acid/0.1% salicylic acid17.0 c,d 9.7 d 9.7 c
            Lely Quaress-Cura1 433% Lactic acid/salicylic acid4 18.9 a,b,c 15.9 a 12.4 b
            Prefoam+1 212% Lactic acid/0.1% salicylic acid18.1 b,c,d 11.7 b,c,d 11.1 b,c
            Virolac Concentrate3 222% Lactic acid/0.1% salicylic acid16.8 c,d 11.2 c,d 10.8 b,c
            Virolac Film1 232% Lactic acid/0.1% Salicylic acid17.2c,d 11.7b,c,d 11.0b,c
            Virolac Spray1 862% Lactic acid/0.1% salicylic acid16.0d 11.3c,d 10.3b,c

            1Ready to use (RTU).

            2Requires activation before use.

            3Concentrate.

            4The concentration of some active ingredients for combination products was not declared by the manufacturer.

            a,b,c,d,e,f,g Inhibitions not sharing the same superscript in a column within an ingredient group were significantly different (P < 0.05).

            # = product number.

            Chlorhexidine

            Twenty-five products belonged to the chlorhexidine group. These products ranged in chlorhexidine concentrations from 0.42% to 1.49% w/w chlorhexidine. For Str. uberis, product 1 (0.74% w/w chlorhexidine) resulted in the largest zone of inhibition of 21.4 mm, which was significantly larger than product 48 (1.49% w/w chlorhexidine), resulting in an inhibition of 15.6 mm (P < 0.05). For Sta. aureus, product 5 (0.5% w/w chlorhexidine) resulted in the largest inhibition of 17.1 mm, which differed significantly from product 82 (0.5% w/w chlorhexidine; 14.0 mm) (P < 0.05). For E. coli, product 2 (0.5% w/w chlorhexidine) resulted in the largest inhibition of 20.6 mm. This differed significantly from product 82, which resulted in a smaller inhibition of 12.3 mm (P < 0.05). Within this ingredient group, a trend was observed for the effectiveness of teat disinfectant products against the three bacterial strains. Product 1 observed the largest inhibition for Str. uberis (21.4 mm) and resulted in the second and third largest inhibitions for Sta. aureus (17.7 mm) and E. coli (18.3 mm), respectively.

            Chlorine dioxide

            The chlorine dioxide ingredient group consisted of five different teat disinfectant products. These products ranged in chlorine dioxide concentrations from 0.0157% to 0.038% w/w. For Str. uberis, Sta. aureus and E. coli, product 95 (0.038% w/w chlorine dioxide) resulted in the largest zones of inhibition of 22.8 mm, 22.4 mm and 21.5 mm, respectively. Furthermore, product 70 (0.05% w/w chlorine dioxide) resulted in a smaller inhibition of 19.4 mm, 18.2 mm and 12.2 mm for Str. uberis, Sta. aureus and E. coli, respectively, compared to product 95 (P < 0.05).

            Diamine

            Only one teat disinfectant product contained the ingredient diamine (0.6% w/w diamine; product 26). This product resulted in zones of inhibition of 16.1 mm, 14.5 mm and 13.6 mm for Str. uberis, Sta. aureus and E. coli, respectively.

            Iodine

            There were 13 iodine products tested within this study. These products ranged from a concentration of 0.25% w/w to 1.35% w/w iodine. For Str. uberis, product 8 (0.5% w/w iodine) resulted in the largest zone of inhibition of 23.0 mm. Product 92 (0.25% w/w iodine) resulted in a smaller inhibition of 12.1 mm, compared to product 9 (P < 0.05). For both Sta. aureus and E. coli, product 91 (1.35% w/w iodine) resulted in the largest inhibition of 18.2 mm and 16.3 mm, respectively. Similar to Str. uberis, product 92 resulted in a smaller inhibition for Sta. aureus (9.2 mm) and E. coli (7.5 mm), which differed significantly from product 91 (P < 0.05). Product 24 (0.5% w/w iodine) resulted in the second largest inhibition for Str. uberis (21.6 mm) and Sta. aureus (17.1 mm) and the fourth largest zone of inhibition for E. coli (12.0 mm).

            Iodine and lactic acid

            A total of five products which contained iodine combined with lactic acid were evaluated. These products ranged in concentrations from 0.15% w/w to 0.5% w/w iodine combined with 0.8% w/w to 1% w/w lactic acid. No significant difference was observed for Str. uberis among the iodine and lactic acid products. However, product 30 (0.5% w/w iodine combined with 0.5% w/w lactic acid) resulted in the numerically largest inhibition of 21.9 mm. Furthermore, product 57 (0.15% w/w iodine combined with 1% w/w lactic acid) resulted in the smallest inhibition (20.3 mm) for Str. uberis. Product 30 resulted in the largest inhibition of 16.5 mm and 12.1 mm against Sta. aureus and E. coli, respectively. Product 57 (0.15% w/w iodine combined with 1% w/w lactic acid) resulted in the smallest inhibition of 13.0 mm and 10.0 mm for Sta. aureus and E. coli, respectively, compared to product 30 (P < 0.05).

            Lactic acid

            Within the study, 15 products which contained various concentrations of lactic acid were tested. These products ranged in lactic acid concentration from 1.76% w/w to 8% w/w lactic acid. For Str. uberis and E. coli, product 94 (4% w/w lactic acid) resulted in the numerically largest inhibition of 22.4 mm and 18.2 mm, respectively. Product 88 (8% w/w lactic acid) showed a significantly smaller inhibition of 16.6 mm for Str. uberis (compared to product 94 [22.4 mm] [P < 0.05]). For E. coli, product 10 (3.6% w/w lactic acid) showed a significantly smaller inhibition of 10.0 mm compared to product 94 (18.2 mm) (P < 0.05). Products 10 and 88 both had the numerically largest inhibition of 19.3 mm for Sta. aureus, which for both products was significantly larger than that for product 96 (5% w/w lactic acid [13.7 mm]) (P < 0.05). Across the lactic acid teat disinfectant products, product 94 resulted in the numerically largest inhibitions against both Str. uberis and E. coli, and the third largest inhibition for Sta. aureus.

            Lactic acid and chlorhexidine

            Of the 96 products tested, 21 of these products contained a combination of lactic acid and chlorhexidine. These products ranged from 1% w/w to 5% w/w lactic acid combined with 0.03% w/w to 1.5% w/w chlorhexidine. Within the lactic acid and chlorhexidine group, product 49 (lactic acid combined with 0.6% w/w chlorhexidine) resulted in the largest inhibitions against Str. uberis, Sta. aureus and E. coli of 22.3 mm, 21.7 mm and 20.3 mm, respectively. Furthermore, product 47 (lactic acid combined with 0.6% w/w chlorhexidine) resulted in the second largest inhibitions for Str. uberis (21.8 mm), Sta. aureus (21.3 mm) and E. coli (18.5 mm) which did not differ significantly from product 49. In comparison to products 49 and 47, product 51 (1% w/w lactic acid combined with 1.49% w/w chlorhexidine) resulted in a smaller inhibition of 16.2 mm for Str. uberis. Alternatively, product 36 (2.5% w/w lactic acid combined with 1.5% w/w chlorhexidine) showed the smallest inhibition for Sta. aureus (15.2 mm) and product 87 (2% w/w lactic acid combined with 0.03% w/w chlorhexidine) showed the smallest inhibition for E. coli (12.2 mm), both of which were significantly different from products 49 and 45 (P < 0.05).

            Lactic acid and hydrogen peroxide

            Only one product contained a combination of the ingredients lactic acid and hydrogen peroxide (product 46; 1.6% w/w lactic acid combined with hydrogen peroxide). This product resulted in inhibitions of 27.9 mm, 25.1 mm and 19.3 mm for Str. uberis, Sta. aureus and E. coli, respectively.

            Lactic acid and salicylic acid

            Within the trial, 10 products contained a combination of lactic acid and salicylic acid. These products ranged from concentrations of 0.25% w/w to 3% w/w lactic acid combined with 0.03% w/w to 0.1% w/w salicylic acid. For Str. uberis, product 27 (0.25% w/w lactic acid combined with salicylic acid) resulted in a significantly greater zone of inhibition of 21.1 mm compared to product 86 (2% w/w lactic acid combined with 0.1% w/w salicylic acid [16.0 mm]) (P < 0.05). For Sta. aureus, product 44 (0.25% w/w lactic acid combined with 0.03% w/w salicylic acid) resulted in the largest zone of inhibition of 16.0 mm. This was significantly different to product 20 (2% w/w lactic acid combined with 0.1% w/w salicylic acid) which had the smallest zone of inhibition of 9.7 mm (P < 0.05). For E. coli, product 53 (0.25% w/w lactic acid combined with salicylic acid) showed the largest zone of inhibition of 16.0 mm, which differed significantly from product 20 with the smallest zone of inhibition of 9.7 mm (P < 0.05).

            While all individual products were not statistically compared, some products were observed to have numerically higher inhibitions against each bacterial strain tested than others within the study. Product 46 was found to result in the largest zones of inhibition for both Str. uberis (27.9 mm) and Sta. aureus (25.1 mm), with product 95 (0.038% w/w chlorine dioxide) achieving the largest zone of inhibition for E. coli (21.5 mm).

            Discussion

            Mastitis control programmes recommend the use of teat disinfection, with some recommending both pre- and post-milking disinfection. By testing teat disinfectant products against bacteria that have previously been identified as the most prevalent mastitis-causing bacteria in Ireland (Keane et al., 2013), the effectiveness of these products can be estimated for use in Ireland. The results of this study show that the range of teat disinfection products showed variation in bacterial inhibition against Str. uberis, Sta. aureus and E. coli, with some individual products and ingredient groups resulting in greater bacterial inhibitions than others.

            The chlorine dioxide ingredient group showed the greatest zones of inhibition for Str. uberis, Sta. aureus and E. coli, which was significantly different to the iodine group for all three bacterial strains. Chlorine dioxide (1%) was previously shown to have large log percentage reductions against Sta. aureus, E. coli and Str. uberis when tested using the excised teat method (Enger et al., 2015). Furthermore, Santos et al. (2016) demonstrated, in vitro, that a 2.5% chlorine dioxide product resulted in reduction levels comparable to a 0.6% iodine product at four different exposure times (15 s, 30 s, 60 s and 300 s) against 50 Sta. aureus strains. However, it has also been stated that chlorine dioxide may be less effective when applied to the teat skin as it can be highly reactive towards organic matter which may be present on the skin surface (Lopes et al., 2012).

            Two ingredient groups within the study contained just one product each; the variation in product numbers within product ingredient groups may represent a limitation within the study. These ingredient groups include diamine (product 26) and lactic acid combined with hydrogen peroxide (product 46). The product containing diamine resulted in small zones of inhibition for all three bacterial strains. This was similar to a previous study where the same diamine product resulted in some of the smallest zones of inhibition against three Sta. aureus isolates, a Str. uberis isolate and an E. coli isolate (Fitzpatrick et al., 2019a). However, when this product was applied to teat skin, it resulted in some of the highest reductions of staphylococcal and streptococcal isolates naturally present on the teat skin (Fitzpatrick et al., 2019b). This could be due to the ingredient diamine being less affected by the presence of organic matter than other ingredients and it is also stable at a wide range of pH (Mondin et al., 2014). This may allow products having this ingredient to be less affected by organic matter on teat skin. In this study, product 46 containing lactic acid combined with hydrogen peroxide had the greatest zones of inhibition for Str. uberis and Sta. aureus. Previously, two hydrogen peroxide products (0.5%) have been shown to achieve a >5 log reduction against Sta. aureus, Str. uberis and E. coli, with these products also being comparable to two chlorine dioxide products (Lopez-Benavides et al., 2012). Furthermore, the use of a hydrogen peroxide product for both pre- and post-milking was shown to reduce the bacterial contamination on teat skin by 65% (Miseikiene et al., 2019). The use of hydrogen peroxide within teat disinfectant products proves to be effective at reducing bacterial load on teat skin but its impact on teat skin condition must be evaluated.

            Additionally, a product containing 0.038% w/w chlorine dioxide (product 95) was found to achieve the greatest zones of inhibition (21.5 mm) against E. coli. Also, a 1% chlorine dioxide product resulted in a large log reduction against an E. coli strain, which was comparable to both a 0.5% and 1% iodophor teat disinfectant, when using the excised teat method (Enger et al., 2015). Furthermore, the use of a chlorine dioxide (0.0157%) product reduced naturally present coliforms on the teat skin by 87.9% (Fitzpatrick et al., 2019b). However, there may be some negative aspects associated with the use of chlorine dioxide products. All chlorine dioxide products used in this study had to be activated/mixed prior to use; also, depending on the product, the time limit for recommended usage after activation ranged from 24 h up to 26 d.

            In the current study, only two ingredient groups showed a trend in effectiveness with increasing concentrations of the active ingredients. These groups included iodine and a combination of iodine and lactic acid. Within the iodine ingredient group, the highest concentration of iodine (1.35% w/w) resulted in the largest zones of inhibition for both Sta. aureus and E. coli, which was significantly different to the lowest concentration of 0.25% w/w iodine, which was also effective for Str. uberis. In the iodine combined with lactic acid group, the concentrations of 0.5% w/w iodine with 0.5% w/w lactic acid and 0.5% w/w iodine with 1% w/w lactic acid resulted in some of the numerically largest inhibitions for all three bacterial strains. A decrease in effectiveness could be observed within these products as iodine levels were reduced. However, products which contained higher concentrations of ingredients, within the other ingredient groups, for example, product 48 (1.49% w/w chlorhexidine), did not always result in the highest level of reduction as would be expected. Limited information with regard to emollient levels in products was available; therefore, the impact of those teat condition agents on the effectiveness of the products could not be evaluated.

            The disc diffusion method used in the current study allows for an effective screening of a large number of teat disinfectant products in a short period of time. However, laboratory methods do not evaluate the true efficacy of a teat disinfectant product. Therefore, further studies must be performed to determine the ability of the products to reduce: (1) bacterial load on the teat skin, (2) new IMIs and (3) impact on teat skin condition.

            Conclusion

            This study has shown that there is a range of alternative teat disinfectant products available which reduce bacterial growth comparable to iodine-based products. The concentration of active ingredient did not influence the effectiveness in the majority of teat disinfectant products. Additionally, different products/ingredients were more effective against specific strains of bacteria within the study. The disc diffusion method is an effective method to screen a large number of teat disinfectant products, but field trials would be required to fully determine the products’ effectiveness in reducing the bacterial load on teat skin and the ability of the products to reduce IMIs.

            Acknowledgements

            The authors gratefully acknowledge funding from Teagasc (project 0006). Sarah Fitzpatrick was in receipt of a Teagasc Walsh Scholarship (Ref: 2016054). Teat disinfection products listed are not an indication of the regulatory status of the products. Check the Department of Agriculture, Food and the Marine (DAFM) Biocidal Products Register (http://www.pcs.agriculture.gov.ie/registers/biocidalproductregisters/) and Health Products Regulatory Authority (HPRA) (http://www.hpra.ie/homepage/veterinary) before purchase.

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            Author and article information

            Journal
            ijafr
            Irish Journal of Agricultural and Food Research
            Compuscript (Ireland )
            2009-9029
            04 June 2021
            : 60
            : 1
            : 78-90
            Affiliations
            [1] 1Teagasc, Animal & Grassland Research and Innovation Centre, Moorepark, Fermoy, County Cork, Ireland
            [2] 2Department of Life Science, Institute of Technology Sligo, County Sligo, Ireland
            Author notes
            †Corresponding author: David Gleeson, E-mail: David.Gleeson@ 123456teagasc.ie
            Article
            10.15212/ijafr-2020-0130
            27547f21-d7b4-437a-8e6a-19f4b8214ceb
            Copyright © 2021 Fitzpatrick, Garvey, Flynn, O’Brien and Gleeson

            This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0).

            Page count
            Figures: 1, Tables: 2, References: 29, Pages: 13
            Product
            Self URI (journal-page): https://www.teagasc.ie/publications/ijafr-journal/
            Categories
            Original Study

            Food science & Technology,Plant science & Botany,Agricultural economics & Resource management,Agriculture,Animal science & Zoology,Pests, Diseases & Weeds
            mastitis,Disc diffusion,teat disinfectant products,screening

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