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

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	7	5% Lactic acid/0.3% chlorhexidine	Pre/post
Arrabawn Udder Guard1	40	0.5% Chlorhexidine	Pre/post
Bacto-Lac1	31	5% Lactic acid/0.05% chlorhexidine	Pre/post
Barri-max1	65	2.4% Lactic acid	Post
Biolac Pre-Post1	44	0.25% Lactic acid/0.03% salicylic acid	Pre/post
Biolac Pre-Post1	59	0.25% Lactic acid/0.03% salicylic acid	Pre/post
Bisept2	70	0.05% Chlorine dioxide	Pre/post
Blue Barrier Spray1	49	Lactic acid/0.6% chlorhexidine3	Post
Blu-gard N Spray1	15	3.46% Lactic acid	Post
C-Dip1	61	0.53% Chlorhexidine	Post
Co-op Source Duo-Teat Shield1	39	2% Lactic acid/0.3% chlorhexidine	Pre/post
D 4 Iodine4	19	0.5% Iodine	Post
Dairy Pro UltraDip1	74	3% Lactic acid	Post
DairyLac SA1	76	3% Lactic acid	Post
Deosan Mastocide1	32	0.5% Chlorhexidine	Post
Deosan Summer Teat Care1	33	0.425% Chlorhexidine	Post
Deosan Super Iodip4	34	0.5% Iodine	Post
Deosan Teat Foam Advance1	13	0.6% Chlorhexidine	Pre/post
Deosan Teatcare Plus1	14	0.425% Chlorhexidine	Post
Deosan Triathalon1	81	1.76% Lactic acid	Pre/post
Dermalac Emprasan1	27	0.25% Lactic acid/salicylic acid3	Pre/post
Dual Dip Supreme1	47	Lactic acid/0.6% chlorhexidine3	Pre/post
Dual Dip1	45	2% Lactic acid/0.3% chlorhexidine	Pre/post
Duo-cel1	38	2.5% Lactic acid/0.3% chlorhexidine	Pre/post
Duogold1	17	2% Lactic acid/0.3% chlorhexidine	Pre/post
Duo-Teat Shield1	25	2% Lactic acid/0.3% chlorhexidine	Pre/post
Emprasan dual1	53	0.25% Lactic acid/salicylic acid3	Pre/post
Flexigard Spray1	94	4% Lactic acid	Post
Fortress Protect Film1	73	3% Lactic acid/0.2% chlorhexidine	Post
Gold Glycodip XL1	62	0.5% Iodine/1% lactic acid	Post
Hamra Red1	12	0.42% Chlorhexidine	Post
Hexa-cel RTU1	42	0.52% Chlorhexidine	Pre/post
Hexaguard1	1	0.74% Chlorhexidine	Pre/post
Hexaklene R1	2	0.5% Chlorhexidine	Pre/post
Hexa-Spray1	82	0.5% Chlorhexidine	Pre/post
Hypraspray1	87	2% Lactic acid/0.03% chlorhexidine	Pre/post
Hypred Quick Spray1	20	2% Lactic acid/0.1% salicylic acid	Pre/post
Ioguard4	3	0.5% Iodine	Post
Ioklar Multi1	92	0.25% Iodine	Pre/post
Io-Shield D1	91	1.35% Iodine	Post
Io-Shield Spray1	93	0.5% Iodine	Post
Kenocidin Spray and Dip1	9	0.5% Chlorhexidine	Post
Kenolac SD1	80	3.6% Lactic acid	Post
Kenolac1	10	3.6% Lactic acid	Post
Kenomint SD1	78	0.5% Chlorhexidine	Post
Kenomint1	77	0.5% Chlorhexidine	Post
Kenomix SD2	89	0.0157% Chlorine dioxide	Post
Kenomix2	11	0.0157% Chlorine dioxide	Post
Kenopure1	79	3.2% Lactic acid	Pre
Lactic Lather1	46	1.6% Lactic acid/hydrogen peroxide3	Pre
Lacto dual1	36	2.5% Lactic acid/1.5% chlorhexidine	Pre/post
Lacto-cel1	35	2.4% Lactic acid	Pre/post
Lacto-Mil1	96	5% Lactic acid	Pre/post
Lactospray1	4	2.4% Lactic acid	Pre/post
Lanodip 4 XL1	30	0.5% Iodine/0.5% lactic acid	Post
Lanodip Pre-Post1	55	0.29% Iodine/0.8% lactic acid	Pre/post
Lely Quaress-Cura1	43	3% Lactic acid/salicylic acid3	Post
Luxdip 50B1	69	0.5% Iodine	Post
Masocare Platinum1	85	0.54% Iodine	Pre/post
Masodine Concentrate4	83	0.5% Iodine	Pre/post
Masodip Platinum1	84	0.436% Chlorhexidine	Pre/post
Maxadine C4	8	0.5% Iodine	Post
Maxidine RTU1	37	0.5% Iodine	Post
Nano Dual1	28	1.93% Lactic acid/0.2% chlorhexidine	Pre/post
Novo Dual1	29	4% Lactic acid/0.27% chlorhexidine	Pre/post
Novodip1	60	4.9% Lactic acid/1.28% chlorhexidine	Post
Novospray1	54	4.9% Lactic acid/0.3% chlorhexidine	Post
Prefoam+1	21	2% Lactic acid/0.1% salicylic acid	Pre
Protect Pre Post1	75	3% Lactic acid/0.25% chlorhexidine	Pre/post
PureChem Chlorhexidine Summer Grade1	52	1.49% Chlorhexidine	Pre/post
PureChem Chlorhexidine1	48	1.49% Chlorhexidine	Post
PureChem Dual Dip1	51	1% Lactic acid/1.49% chlorhexidine	Pre/post
PureChem Iodophor4	50	0.5% Iodine	Post
Quatro1	41	0.5% Chlorhexidine	Pre/post
SalvoDip B1	71	2.4% Lactic acid	Post
Salvohex1	67	2% Lactic acid/0.3% chlorhexidine	Post
Salvospray1	68	2.4% Lactic acid	Pre/post
SensoDip 501	16	0.5% Chlorhexidine	Post
SensoDip1	72	0.5% Chlorhexidine	Pre/post
Sensospray1	66	0.5% Chlorhexidine	Post
Silkdip4	24	0.5% Iodine	Post
Summer C-Dip1	58	0.5% Chlorhexidine	Post
Super Cow Teat Foam1	26	0.6% Diamine	Pre/post
Supergold1	18	0.5% Chlorhexidine	Pre/post
Supreme1	64	2.5% Lactic acid/0.37% chlorhexidine	Pre/post
Sure Spray Duo1	6	2% Lactic acid/0.3% chlorhexidine	Pre/post
Surespray1	5	0.5% Chlorhexidine	Pre/post
Synofilm1	88	8% Lactic acid	Post
Teat Gard C1	63	0.5% Chlorhexidine	Pre/post
TriCide Gold1	57	0.15% Iodine/1% lactic acid	Pre/post
TriCide1	56	0.15% Iodine/1% lactic acid	Post
Uddergold2	90	0.32% Acidified sodium chlorite	Post
Valiant2	95	0.038% Sodium chloride	Post
Virolac Film1	23	2% Lactic acid/0.1% salicylic acid	Post
Virolac Spray1	86	2% Lactic acid/0.1% salicylic acid	Pre/post
Virolac Concentrate4	22	2% Lactic acid/0.1% salicylic acid	Pre/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	40	0.5% Chlorhexidine	16.6 d,e,f,g	15.3 b,c,d	16.2 b,c,d
C-Dip1	61	0.53% Chlorhexidine	17.6 d,e,f,g	15.7 b,c,d	15.6 b,c,d,e
Deosan Mastocide1	32	0.5% Chlorhexidine	19.1 a,b,c,d,e,f	17.4 a,b,c	16.7 b,c,d
Deosan Summer Teat Care1	33	0.425% Chlorhexidine	17.4 d,e,f,g	16.7 a,b,c,d	17.9 a,b,c,d
Deosan Teat Foam Advance1	13	0.6% Chlorhexidine	19.5 a,b,c,d,e	16.1 a,b,c,d	16.3 b,c,d
Deosan Teatcare Plus1	14	0.425% Chlorhexidine	15.8 g	16.9 a,b,c,d	17.6 a,b,c,d
Hamra Red1	12	0.42% Chlorhexidine	17.8 b,c,d,e,f,g	16.0 b,c,d	15.9 b,c,d,e
Hexa-cel RTU1	42	0.52% Chlorhexidine	15.9 f,g	16.1 a,b,c,d	15.1 b,c,d,e
Hexaguard1	1	0.74% Chlorhexidine	21.4 a	17.7 a,b	18.3 a,b,c
Hexaklene R1	2	0.5% Chlorhexidine	20.9 a,b,c	15.9 b,c,d	20.6 a
Hexa-Spray1	82	0.5% Chlorhexidine	17.3 d,e,f,g	14.0 d	12.3 e
Kenocidin Spray/Dip1	9	0.5% Chlorhexidine	17.0 d,e,f,g	16.2 a,b,c,d	14.2 d,e
Kenomint1	77	0.5% Chlorhexidine	17.7 c,d,e,f,g	16.0 b,c,d	16.3 b,c,d
Kenomint SD1	78	0.5% Chlorhexidine	19.1 a,b,c,d,e,f	14.6 c,d	14.5 c,d,e
Masodip Platinum1	84	0.436% Chlorhexidine	16.4 e,f,g	17.9 a,b	14.4 d,e
PureChem Chlorhexidine1	48	1.49% Chlorhexidine	15.6 g	15.0 b,c,d	14.8 b,c,d,e
PureChem Chlorhexidine Summer Grade1	52	1.49% Chlorhexidine	17.4 d,e,f,g	16.3 a,b,c,d	16.3 b,c,d
Quatro1	41	0.5% Chlorhexidine	16.1 f,g	16.6 a,b,c,d	16.5 b,c,d
SensoDip1	72	0.5% Chlorhexidine	17.0 d,e,f,g	15.9 b,c,d	15.9 b,c,d,e
SensoDip 501	16	0.5% Chlorhexidine	18.4 a,b,c,d,e,f,g	17.4 a,b,c	17.3 a,b,c,d
Sensospray1	66	0.5% Chlorhexidine	16.9 d,e,f,g	17.1 a,b,c,d	14.9 b,c,d,e
Summer C-Dip1	58	0.5% Chlorhexidine	19.4 a,b,c,d,e,	15.1 b,c,d	14.5 c,d,e
Supergold1	18	0.5% Chlorhexidine	21.0 a,b	16.5 a,b,c,d	18.5 a,b
Surespray1	5	0.5% Chlorhexidine	19.8 a,b,c,d	19.2 a	18.2 a,b,c
Teat Gard C1	63	0.5% Chlorhexidine	16.0 f,g	15.2 b,c,d	15.4 b,c,d,e
Chlorine dioxide products
Bisept2	70	0.05% Chlorine dioxide	19.4 c	18.5 b	12.2 c
Kenomix2	11	0.0157% Chlorine dioxide	22.6 a	18.2 b	19.3 a,b
Kenomix SD2	89	0.0157% Chlorine dioxide	21.0 b	21.4 a,b	20.5 a,b
Uddergold2	90	0.32% Acidified sodium chlorite	20.9 b,c	19.5 a,b	17.3 b
Valiant2	95	0.038% Sodium chloride	22.8 a	22.4 a	21.5 a
Diamine products
Super Cow Teat Foam1	26	0.6% Diamine	16.1	14.5	13.6
Iodine products
D 4 Iodine3	19	0.5% Iodine	21.4 a,b,c	16.1 a,b,c	10.9 b
Deosan Super Iodip3	34	0.5% Iodine	20.0 b,c,d,e	15.2 b,c,d,e	11.7 b
Ioguard3	3	0.5% Iodine	21.0 a,b,c,d	13.5 c,d,e	10.2 b,c
Ioklar Multi1	92	0.25% Iodine	12.1 g	9.2 f	7.5 c
Io-Shield D1	91	1.35% Iodine	19.0 d,e,f	18.2 a	16.3 a
Io-Shield Spray1	93	0.5% Iodine	17.3 f	14.5 b,c,d,e	12.8 b
Luxdip 50B1	69	0.5% Iodine	19.2 c,d,e,f	13.7 c,d,e	11.4 b
Masocare Platinum1	85	0.54% Iodine	19.3 c,d,e,f	14.5 b,c,d,e	12.4 b
Masodine Concentrate3	83	0.5% Iodine	20.7 a,b,c,d,e	15.5 b,c,d	10.8 b
Maxadine C3	8	0.5% Iodine	23.0 a	15.8 a,b,c,d	11.9 b
Maxidine RTU1	37	0.5% Iodine	18.9 d,e,f	13.3 d,e	11.4 b
PureChem Iodophor3	50	0.5% Iodine	18.6 e,f	12.6 f	10.5 b
Silkdip3	24	0.5% Iodine	21.6 a,b	17.1 a,b	12.0 b
Iodine and lactic acid products
Gold Glycodip XL1	62	0.5% Iodine/1% lactic acid	21.6 a	15.3 a,b	12.0 a
Lanodip 4 XL1	30	0.5% Iodine/0.5% lactic acid	21.9 a	16.5 a	12.1 a
Lanodip Pre-Post1	55	0.29% Iodine/0.8% lactic acid	21.1 a	13.3 b,c	10.1 b
TriCide1	56	0.15% Iodine/1% lactic acid	21.1 a	13.0 c	10.0 b
TriCide Gold1	57	0.15% Iodine/1% lactic acid	20.3 a	13.2 c	10.1 b
Lactic acid products
Barri-max1	65	2.4% Lactic acid	19.7 b,c,d	16.3 a,b,c	13.6 b
Blu-gard N Spray1	15	3.46% Lactic acid	19.9 b,c,d	15.6 b,c	11.4 b,c,d
Dairy Pro UltraDip1	74	3% Lactic acid	17.1 e,f	16.1 a,b,c	12.1 b,c,d
DairyLac SA1	76	3% Lactic acid	18.8 d,e,f	14.5 c	11.8 b,c,d
Deosan Triathalon1	81	1.76% Lactic acid	19.7 b,c,d	14.2 c	11.4 b,c,d
Flexigard Spray1	94	4% Lactic acid	22.4 a	19.2 a	18.2 a
Kenolac1	10	3.6% Lactic acid	22.3 a	19.3 a	10.0 d
Kenolac SD1	80	3.6% Lactic acid	19.a c,d,e	15.0 b,c	13.2 b,c
Kenopure1	79	3.2% Lactic acid	21.6 a,b	16.4 a,b,c	11.2 b,c,d
Lacto-cel1	35	2.4% Lactic acid	19.9 b,c,d	15.9 b,c	12.1 b,c,d
Lacto-Mil1	96	5% Lactic acid	19.1 c,d,e	13.7 c	10.6 c,d
Lactospray1	4	2.4% Lactic acid	21.3 a,b,c	19.2 a	10.2 d
SalvoDip B1	71	2.4% Lactic acid	19.6 b,c,d	16.6 a,b,c	11.5 b,c,d
Salvospray1	68	2.4% Lactic acid	18.8 d,e,f	17.8 a,b	11.5 b,c,d
Synofilm1	88	8% Lactic acid	16.6 f	19.3 a	17.3 a
Lactic acid and chlorhexidine products
Arkshield1	7	5% Lactic acid/0.3% chlorhexidine	21.6 a,b,c	18.9 a,b,c	15.4 b,c,d
Bacto-Lac1	31	5% Lactic acid/0.05% chlorhexidine	18.8 c,d,e,f	15.9 d,e	14.1 c,d
Blue Barrier Spray1	49	Lactic acid/0.6% chlorhexidine4	22.3 a	21.7 a	20.3 a
Co-op Source Duo-Teat Shield1	39	2% Lactic acid/0.3% chlorhexidine	18.1 d,e,f	16.5 c,d,e	15.4 b,c,d
Dual Dip1	45	2% Lactic acid/0.3% chlorhexidine	18.2 d,e,f	16.0 d,e	15.0 b,c,d
Dual Dip Supreme1	47	Lactic acid/0.6% Chlorhexidine4	21.8 a,b	21.3 a,b	18.5 a,b
Duo-cel1	38	2.5% Lactic acid/0.3% chlorhexidine	18.5 d,e,f	16.4 c,d,e	16.2 b,c
Duogold1	17	2% Lactic acid/0.3% chlorhexidine	19.7 a,b,c,d,e	16.8 c,d,e	17.3 a,b,c
Duo-Teat Shield1	25	2% Lactic acid/0.3% chlorhexidine	17.9 d,e,f	16.9 c,d,e	16.8 a,b,c
Fortress Protect Film1	73	3% Lactic acid/0.2% chlorhexidine	20.3 a,b,c,d,e	17.6 c,d,e	13.9 c,d
Hypraspray1	87	2% Lactic acid/0.03% chlorhexidine	19.1 b,c,d,e	16.7 c,d,e	12.2 d
Lacto dual1	36	2.5% Lactic acid/1.5% chlorhexidine	18.2 d,e,f	15.2 e	16.6 b,c
Nano Dual1	28	1.93% Lactic acid/0.2% chlorhexidine	21.6 a,b,c	16.9 c,d,e	14.8 c,d
Novo Dual1	29	4% Lactic acid/0.27% chlorhexidine	20.8 a,b,c,d	17.5 c,d,e	15.6 b,c,d
Novodip1	60	4.9% Lactic acid/1.28% chlorhexidine	19.3 b,c,d,e	19.1 a,b,c	16.0 b,c
Novospray1	54	4.9% Lactic acid/0.3% chlorhexidine	20.0 a,b,c,d,e	18.4 b,c,d	16.3 b,c
Protect Pre Post1	75	3% Lactic acid/0.25% chlorhexidine	21.7 a,b	18.2 c,d	14.0 c,d
PureChem Dual Dip1	51	1% Lactic acid/1.49% chlorhexidine	16.2 f	16.3 c,d,e	14.5 a
Salvohex1	67	2% Lactic acid/ 0.3% chlorhexidine	17.8 e,f	16.2 c,d,e	16. 8 a,b,c
Supreme1	64	2.5% Lactic acid/0.375% chlorhexidine	19.2 b,c,d,e	16.8 c,d,e	15.4 b,c,d
Sure Spray Duo1	6	2% Lactic acid/0.3% chlorhexidine	18.6 d,e,f	17.2 c,d,e	15.4 b,c,d
Lactic acid and hydrogen peroxide products
Lactic Lather1	46	1.6% Lactic acid/ hydrogen peroxide4	27.9	25.1	19.3
Lactic acid and salicylic acid products
Biolac Pre-Post1	44	0.25% Lactic acid/0.03% salicylic acid	19.7 a,b	16.0 a	11.4 b
Biolac Pre-Post1	59	0.25% Lactic acid/0.03% salicylic acid	21.0 a	15.1 a	11.8 b,c
Dermalac Emprasan1	27	0.25% Lactic acid/salicylic acid4	21.1 a	14.3 a,b,c	11.0 b,c
Emprasan dual1	53	0.25% Lactic acid/salicylic acid4	19.6 a,b	14.8 a,b	16.0 a
Hypred Quick Spray1	20	2% Lactic acid/0.1% salicylic acid	17.0 c,d	9.7 d	9.7 c
Lely Quaress-Cura1	43	3% Lactic acid/salicylic acid4	18.9 a,b,c	15.9 a	12.4 b
Prefoam+1	21	2% Lactic acid/0.1% salicylic acid	18.1 b,c,d	11.7 b,c,d	11.1 b,c
Virolac Concentrate3	22	2% Lactic acid/0.1% salicylic acid	16.8 c,d	11.2 c,d	10.8 b,c
Virolac Film1	23	2% Lactic acid/0.1% Salicylic acid	17.2c,d	11.7b,c,d	11.0b,c
Virolac Spray1	86	2% Lactic acid/0.1% salicylic acid	16.0d	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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