Survival of intestinal spirochaete strains from chickens in the presence of disinfectants and in faeces held at different temperatures

Abstract

This study aimed to evaluate the efficacy of some commonly used disinfectants in inactivating the pathogenic avian intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli, and to examine spirochaete survival in chicken caecal faeces held at 4°C, 25°C or 37°C. Six disinfectants were evaluated at their recommended working concentrations: alkaline salts, quaternary ammonium, iodine as an iodophor, chlorine from a chlorine-release agent, glutaraldehyde and hydrogen peroxide. All but alkaline salts inactivated two different concentrations of both spirochaete species in less than 1 min in the presence of organic matter. Both spirochaete species at three different cell concentrations survived in caecal faeces at 37°C for between 2 and 17 h. B. intermedia tended to survive for longer than B. pilosicoli, but the maximum survival time for both species at 4°C was only 72 to 84 h. Hence, avian intestinal spirochaetes are rapidly inactivated by several common disinfectants, and their survival time in chicken caecal faeces is much less than has been reported for porcine intestinal spirochaetes in porcine faeces. It should be relatively easy to break the cycle of infection between batches of laying birds by resting sheds for a few days, and by using disinfectants on any residual faecal matter.

1 Introduction

Avian intestinal spirochaetosis is a disease of commercial layer and meat breeder chickens resulting from colonization of the caeca and colon by anaerobic intestinal spirochaetes (Davelaar et al., 1986; Swayne, 1997; Stephens & Hampson, 2001). The causal bacteria, formerly placed in the genus Serpulina, have been reassigned to the genus Brachyspira. At least seven species colonize poultry, but only three, Brachyspira intermedia, Brachyspira pilosicoli and Brachyspira alvinipulli, are currently considered to be pathogenic in chickens (McLaren et al., 1997). In Australia, B. intermedia and B. pilosicoli are the two main pathogenic species, with both being present in about 10% of layer and broiler breeder flocks (Stephens & Hampson, 1999).

Avian intestinal spirochaetosis has been associated with delayed and/or reduced egg production, and chronic diarrhoea in adult birds (Griffiths et al., 1987; Dwars et al., 1992; Swayne et al., 1992; Trampel et al., 1994; Swayne et al., 1995). The diarrhoea leads to faecal staining of eggs, and the resultant wet litter presents problems in cage cleaning, odour emission and attraction of flies. Broiler chicks hatched from eggs of infected parents have a worse performance than those of uninfected parents (Dwars et al., 1993; Smit et al., 1998).

Relatively few studies have been carried out on these organisms, or on the control of the associated disease(s). In particular, there is a lack of basic data about the likely survival time of these organisms in chicken faeces, as well as the effectiveness of some common disinfectants that might be used to remove them from the environment of poultry sheds. The current study was undertaken to provide this information.

2 Materials and Methods

2.1 Spirochaete strains and culture techniques

The two pathogenic avian spirochaete strains used were B. intermedia strain HB60 (Hampson & McLaren, 1999) and B. pilosicoli strain CPSp1 (Stephens & Hampson, 2002). Both were obtained from the culture collection held at the Reference Centre for Intestinal Spirochaetes at Murdoch University. They were grown to mid-log phase in Kunkle's pre-reduced anaerobic broth (Kunkle et al., 1986), and enumerated using a counting chamber.

2.2 Survival in the presence of disinfectants

Survival of the two spirochaete strains was tested in the presence of six disinfectants commonly used in poultry houses. These were alkaline salts (Active Dot; Peerless Emulsion Products Pty Ltd, Australia), quaternary ammonium (Quickmaster Lemon; Kwikmaster Products, Australia), iodine as an iodophor (Vetadine; PharmTech Pty Ltd, Australia), chlorine from Chloramine T (Halamid; Akzo Nobel Chemicals, The Netherlands), glutaraldehyde (Sigma, USA), and hydrogen peroxide (BDH, Australia). The disinfectants were tested at the concentration recommended by the manufacturers (Table 1). For each disinfectant, two cell concentrations of each spirochaete strain were tested (10⁸ and 10⁶ cells/ml) at four different exposure time intervals (1, 5, 10 and 30 min). Control strains grown in the absence of disinfectant were also included, and these were handled in the same way as for the treated samples. The spirochaetes were thoroughly mixed with the respective disinfectants in 10 ml anaerobic Trypticase Soy broth containing 10% foetal calf serum. One millilitre volumes of the broths containing the spirochaetes were then removed after the set time intervals, and added to 10 ml fresh broth. After mixing, these tubes were centrifuged at 2500 g for 15 min, and the supernatant containing any residual disinfectant discarded. A swab was then placed in the pellet and this was plated to Trypticase Soy Agar containing 5% defibrinated ovine blood, and incubated anaerobically (94% H₂ and 6% CO₂) at 37°C for 7 days. Subsequently, growth on the plates was examined, scraped off, and observed under a phase contrast microscope.

Survival time of Brachyspira intermedia and Brachyspira pilosicoli strains at two cell concentrations in the presence of six disinfectants

		Survival time (min)^a
		B. intermedia		B. pilosicoli
Disinfectant active constituent (stock concentration)	Working dilution in broth	10^8 cells/ml	10^6 cells/ml	10^8 cells/ml	10^6 cells/ml
Alkaline salts (61 g/l)	1:25	>10 to<30	>1 to<5	>1 to<5	<1
Quaternary ammonium (15 g/l)	1:20	<1	<1	<1	<1
Iodine (active) (16 g/l)	1:167	<1	<1	<1	<1
Chlorine (250 g/kg)	1:200	<1	<1	<1	<1
Glutaraldehyde (25% v/v)	1:25	<1	<1	<1	<1
Hydrogen peroxide (30% v/v)	1:30	<1	<1	<1	<1
^a Both species at both concentrations still viable in the control broths after 30 min.

2.3 Survival in faeces at different temperatures

Chicken caecal faeces that were tested negative for intestinal spirochaetes by culture, and also by B. pilosicoli 16S rDNA polymerase chain reaction (La et al., 2003) and B. intermedia NADH oxidase polymerase chain reaction (Atyeo et al., 1999) of growth on the primary plate, were pooled, thoroughly mixed and then divided into 20 g aliquots. Samples were seeded with different concentrates of either B. intermedia or B. pilosicoli cells (10⁵, 10⁷ and 10⁹ cells/g faeces), or with an equivalent volume of sterile broth, and were thoroughly mixed in a blender. These samples were held in sealed containers at either 4°C, 25°C or 37°C, and cotton-tipped bacteriology swabs were inserted into these at times 0 and 2 h, and then once every 9 to 15 h until 14 days had elapsed. The swabs were plated to a selective agar for isolation of intestinal spirochaetes, consisting of Trypticase Soy agar containing 400 μg/ml spectinomycin and 25 μg/ml each of colistin and vancomycin (Jenkinson & Wingar, 1981). Plates were incubated at 37°C in an anaerobic jar in an atmosphere of 94% H₂ and 6% CO₂ for 7 days, and spirochaete growth on the plate was recorded as being either present or absent.

3 Results

The results for survival times of B. intermedia and B. pilosicoli in the presence of the disinfectants are presented in Table 1. Both species were still viable in the control broths without disinfectants after 30 min. The two species showed similar sensitivities to the disinfectants, except that the B. pilosicoli strain was slightly more susceptible to the alkaline salts. Except in the case of the alkaline salts, increasing the cell concentration did not increase the survival time. Apart from alkaline salts, the other five disinfectants all inactivated the spirochaetes in less than 1 min.

The survival times for B. intermedia and B. pilosicoli cells held in chicken caecal faeces at different temperatures are presented in Table 2. The two species had broadly similar survival times, although both concentrations of B. pilosicoli cells survived for a slightly shorter period than the B. intermedia cells at 25°C, and survived for a shorter time at 10⁵ cells/g when held at 4°C.

Survival time of three different concentrations of Brachyspira intermedia or Brachyspira pilosicoli cells suspended in 20 g chicken caecal faeces held at three different temperatures

	Survival time (h)
	B. intermedia			B. pilosicoli
Temperature (°C)	10^5/g	10^7/g	10^9/g	10^5/g	10^7/g	10^9/g
4	>60 to<72	>72 to<84	>72 to<84	>41 to<50	>72 to<84	>72 to<84
25	>50 to<59	>59 to<74	>59 to<74	>17 to<26	>41 to<50	>41 to<50
37	>2 to<17	>2 to<17	>2 to<17	>2 to<17	>2 to<17	>2 to<17

The survival time for both species was considerably reduced at 37°C compared with at the two other temperatures, being only between 2 and 17 h, and was longer at 4°C than at 25°C. Survival times for both species at 4°C and 25°C, but not at 37°C, tended to increase slightly at the higher cell concentrations. The maximum survival time for both species was between 72 and 84 h at higher cell concentrations in faeces held at 4°C.

4 Discussion

This study indicates that pathogenic intestinal spirochaetes infecting chickens are relatively susceptible to disinfectants in common use, even in the presence of organic matter. The related intestinal spirochaete Brachyspira hyodysenteriae, the agent of swine dysentery, has similarly been shown to be rapidly inactivated in a faecal slurry by a number of common disinfectant solutions (Chia & Taylor, 1978). In the current study, the disinfectants were tested against the spirochaetes in Trypticase Soy broth containing 10% serum, with this medium acting as a standard controlled source of organic matter. Disinfectants are normally inactivated by contact with organic matter, hence they should be tested in the presence of sources of this material to evaluate their probable efficacy under field conditions. The use of a broth medium, which is rich in organic material, has been recommended for use when testing disinfectants against other avian pathogenic bacteria (Ruano et al., 2001). The alternative of testing disinfectant efficacy on faeces seeded with bacteria can be problematic, since it is difficult to remove residual disinfectants when transferring the bacteria to suitable growth medium for subsequent evaluation of their viability. Despite using a standardized broth method of evaluation in this study, the possibility remains that spirochaetes could have survived for longer if they were in the centre of a faecal bolus, where active disinfectants could not penetrate.

Both species of spirochaete were relatively short-lived in chicken caecal faeces. Previous studies on intestinal spirochaete survival have concentrated on species isolated from pigs, particularly B. hyodysenteriae. Survival in these studies has generally been much longer than the survival times found here. For example, Chia & Taylor (1978) showed that B. hyodysenteriae survived for 48 days in dysenteric faeces held between 0 and 10°C, although it only survived for 7 days at 25°C, and less than 24 h at 37°C. In another study, B. hyodysenteriae survived for 10 days in soil held at 10°C, but this increased to 78 days in soil in the presence of 10% pig faeces, and was 112 days in pure pig faeces (Boye et al., 2001). In the same study, B. pilosicoli was found to be more resistant, surviving for 119 days in pure soil, and 210 days both in soil with 10% pig faeces and in pure pig faeces. Oxberry et al. (1998) also found B. pilosicoli to be relatively resistant, surviving in lake water at 4°C for 66 days, although its survival was reduced to 4 days at 25°C. The results from the current study confirm that both B. pilosicoli and B. intermedia survive for shorter periods at warmer than colder temperatures. Overall, their survival times in chicken caecal faeces was greatly reduced compared with the survival time of spirochaetes held in pig faeces, or in water, being less than 84 h even at 4°C. Chicken faeces tend to be acidic and relatively dry, and this adverse environment may be responsible for the short survival times. It is possible that the methodology used for seeding faeces with spirochaetes may have affected the viability of the organisms, and the distribution of spirochaetes through the seeded faeces was unlikely to be completely representative of the distribution in the faeces of naturally infected birds. In the latter, it is probable that there are accumulations of spirochaetal cells in parts of the faeces, and these may survive for longer than cells homogeneously distributed through seeded faeces.

The practical implications of the findings of the study are that avian intestinal spirochaetes are likely to be relatively fragile in the environment of a chicken shed. It should be easy to break cycles of infection between batches of layer birds by resting sheds and equipment for only a few days, and applying common disinfectants to contaminated equipment after cleaning to remove organic matter. The results also suggest that where infection occurs in new batches of birds, it is unlikely to arise from spirochaetes from a previous batch of birds surviving in the shed. Infection is more likely to arise by cross-contamination from sheds of older birds on the same site, or from exogenous sources such as the faeces of wild birds.

Acknowledgments

This study was supported by a grant from the Australian Chicken Meat and Egg Councils, administered through the Australian Rural Industries Research and Development Corporation.