Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Detection of methane and quantification of methanogenic archaea in faeces from young broiler chickens using real-time PCR S. Saengkerdsub1, P. Herrera1, C.L. Woodward1, R.C. Anderson2, D.J. Nisbet2 and S.C. Ricke1 1 Department of Poultry Science, Texas A & M University, College Station, TX, USA 2 United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, TX, USA
Keywords avian, broiler, faeces, methanogen, real-time polymerase chain reaction. Correspondence Steven C. Ricke, Department of Food Science, University of Arkansas, 2650 N., Young Ave, Fayetteville, AR 72704, USA. E-mail:
[email protected]" Present address Paul Herrera and Steven C. Ricke, Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA.
2006 ⁄ 0406: received 22 March 2007, revised 4 May 2007 and accepted 16 July 2007 doi:10.1111/j.1472-765X.2007.02243.x
Abstract Aims: To detect the presence of methanogens in the faeces of broiler chicks during the first 2 weeks of age. Methods and Results: Chicken faecal samples from 120 broiler chicks were incubated for methane gas formation and methanogenic archaea were analysed using real-time PCR. The copy number of the order Methanobacteriales 16S rDNA gene in chicken faeces when the broilers were 3–12 days of age, litter and house flies collected in the bird house ranged from 4Æ19 to 5Æ51 log10 g)1 wet weight. The number of positive methane culture tubes increased from 25% to 100% as the birds aged. Conclusions: Methanogens were successfully detected in faecal samples from 3- to 12-day-old broilers, as well as litter and house flies using real-time PCR. The copy number of methanogenic 16S rDNA gene in these samples was also similar to the number observed in litter and house flies. Significance and Impact of the Study: The same methanogens consistently appeared in chicken faeces a few days after birth. Detection of the methanogenic bacteria in litter and house flies implicated them as potential environmental sources for methanogen colonization in broiler chicks.
Introduction The complex microbial community that inhabits the gastrointestinal tract of poultry plays an important role in the health and well-being of the host and serves as a barrier to colonization by foodborne pathogens (Patterson and Burkholder 2003). When chicks are hatched, the intestinal tract is sterile and is successively colonized by various micro-organisms from the hen and the surrounding environment (Conway 1997). Barnes et al. (1972) and Salanitro et al. (1974) observed that the microbial community structure in chicken caeca varies with age. In chickens, the gastrointestinal tract becomes rapidly colonized by bacteria, with maximum bacterial densities achieved within the first 5 days after hatching. During the following weeks, the composition of microflora changes markedly (Apajalahti 2005). Recently, molecular
approaches have provided ways to directly observe microbial diversity in the gastrointestinal tract without culture. Hume et al. (2003), using DGGE (denaturing gradient gel electrophoresis) analysis, found eight and 26 major bands from chicken caecal samples at 2 and 32 days of age. By constructing 16S rDNA clone libraries, some anaerobic bacteria, such as Bacteroides spp., Clostridium spp. and Ruminococcus spp., were found in the caeca and ilea when the chicks were 3 days of age (Lu et al. 2003a). In most animals, methanogens become established in the intestinal tract relatively early (Maczulak et al. 1989; Morvan et al. 1994; Zhu and Joerger 2003; Skillman et al. 2004). Methanogen densities reach 104 and 109 per gram in rumen fluid of grazing lambs at 1 and 3 weeks of age, respectively (Skillman et al. 2004). Morvan et al. (1994) found that methanogens colonized the rumen of lambs 30 h after birth and reached 106 ml)1 at 15 days. In the
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rat, the concentration of methanogens increases from 105 per gram dry weight at 3 weeks of age (shortly after weaning) to 109 per gram dry weight at 96 weeks of age (shortly before the end of the life span) (Maczulak et al. 1989). Rutili et al. (1996) observed that methanogenic bacteria were not detected in faecal samples obtained from children under 27 months, but were detected in 40% and 60% of faecal samples from 3- to 5-year-old children, respectively. Based on a variety of studies, methanogens are thought to be indigenous in the adult chicken gastrointestinal tract (Ricke et al. 2004). Our previous work demonstrated that methane production yielded approx. 11 lmol g)1 during in vitro 24-h incubation of adult laying hen caecal contents (Saengkerdsub et al. 2006). In contrast to other anaerobes in the chicken caeca, studies identifying when colonization occurs and the quantity of methanogens in chickens are limited. One in vitro caeca study observed methane gas production when the chicks were 2 months old (Marounek and Rada 1998). However, Zhu and Joerger (2003) using a fluorescent in situ hybridization (FISH) method found that methanogenic bacteria became established in caeca of very young chicks. In this study, we describe methanogen detection and quantification in the faeces of broiler chicks that were fed a corn-soy diet during the first 2 weeks of age. Materials and methods Sample preparation A total of 121-day-old commercial broiler chicks were placed on sawdust, which had been previously used as bedding by laying hens. These chicks were maintained on the Texas A&M University (TAMU, College Station, TX, USA) layer feed ration which was composed of (%): corn 56Æ72; soya bean meal 31Æ63; vegetable oil 7Æ68; monocalcium phosphate 1Æ69; calcium carbonate 1Æ56; methionine (98%) 0Æ17; vitamin premix 0Æ25; NaCl 0Æ25 and trace mineral premix 0Æ05. The chicks were divided into 12 groups of 10 chicks apiece. On days 3 and 4, faecal samples from groups 3, 5 and 7 were collected for methanogen detection using real-time PCR. On days 5, 9 and 12, faeces from all 12 groups were collected and subjected to both bacteriological cultivation for the detection of methane production and real-time PCR for the quantification of methanogenic bacteria. Houseflies (Musca domestica) and litter from the bird house were collected to quantify methanogenic bacteria and to detect methane production using the same methods as the faecal samples. Dust and layer feed ration were also collected for bacterial cultivation. 630
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DNA extraction Bacterial genomic DNA was isolated by the method of Wright et al. (1997) with some modifications. Faecal samples were suspended in Tris-EDTA (TE) buffer and treated with proteinase K for 1 h at 37C, followed by five cycles of freezing at )80C for 1 h and heating in a water bath at 65C for 30 min. The lysate was treated with cetryltrimethylammonium bromide ⁄ sodium chloride (CTAB ⁄ NaCl). The CTAB was extracted with an equal volume of chloroform–isoamyl alcohol (24 : 1), mixed and centrifuged at 7000 g for 5 min. The DNA solution was transferred to a new microcentrifuge tube with an equal volume of phenol–chloroform–isoamyl alcohol (25 : 24 : 1), mixed and centrifuged at 7000 g for 5 min and precipitated with isopropanol. The extracted DNA was further purified with a Dneasy Tissue kit (Quigen, Valencia, CA, USA). The DNA solution was stored at )20C until used in the PCR assay. Quantitative PCR assays Calibration standards for the quantitative PCR assays were developed with a 10-fold dilution series of a plasmid containing sequence CH101 closely related to Methanobrevibacter woesei GS (accession number U55237). Plasmid copy number was calculated from plasmid molecular weight, and plasmid concentration was measured with Picogreen (Molecular Probes, Eugene, OR, USA) using a Spectrafluor Plus (Research Triangle Park, Raleigh, NC, USA). The plasmid stock solution was kept at )20C at a concentration of 1012 copies number ml)1. The order Methanobacteriales-specific forward, Taqman, reverse primers MBT857F, MBT929F and MBT1196R (Yu et al. 2005) were used for real-time PCR. The quantitative PCR were performed in triplicate using the reaction conditions described in Yu et al. (2005). Methanogenesis detection Faecal samples were transferred to an anaerobic glove box (Coy Laboratory Products, Grass Lake, MI, USA) and maintained in an atmosphere of 95% N2 ⁄ 5% H2. The faecal samples were added into a serum tube contained 9 ml of BRN medium (Balch et al. 1979; Miller and Wolin 1982). The tubes were removed from the glove box after being sealed with rubber stoppers and aluminium caps. Each tube was flushed with 80% H2 ⁄ 20% CO2 under 200 kPa. The bottles were incubated standing at 37C and mixed manually once per day. After 20 days, methane concentrations in the tubes’ headspace were measured by gas chromatography (SRI, model 8610C, Torrance, CA, USA). Tubes with methane concentrations >100 ppm (lg ml)1)
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Threshold cycle (C T)
30 25 20 15 10 5 0
R 2 = 0·99 3
4
5
6 7 8 9 10 Log10 gene copies (ml–1)
11
12
13
Figure 1 Standard curve generated by analysis of a dilution series of the plasmid containing sequence CH101. The clone was the predominant methanogenic ribotype from our previous study (Saengkerdsub et al. 2007). The mean values and standard deviation of log10 16S rDNA copy numbers were performed from triplicates within the same run.
were considered positive for the presence of methanogenic bacteria in faecal samples. Results In this study, quantification of methanogens in the chick faecal samples was performed using real-time PCR. As the predominant methanogenic 16S rDNA sequence isolated from chicken caeca was previously determined to be related to M. woesei (Saengkerdsub et al. 2007) belonging to the order Methanobacteriales, the copy number of methanogenic 16S rDNA present in samples was
Table 1 The log10 copy numbers of 16S rDNA methanogenic archaea in chicken faecal samples
measured by using primers MBT specifically designed for the order Methanobacteriales (Yu et al. 2005). The plasmid containing 16S rDNA gene of the primary phylotype CH101 in chicken caeca was used as the standard in the PCR assay. The details of the methanogenic 16S rDNA sequences found in the chicken caeca will be discussed elsewhere. A standard curve was performed to plot the threshold cycle number (CT) against the plasmid copy number. The concentrations of plasmid were prepared from 10-fold serial dilutions of the stock plasmid solution. As illustrated in Fig. 1, the CT value is linearly proportional to the plasmid copy number of the with square regression coefficient of ‡0Æ99 over a range of log10 3Æ0– 12Æ0 gene copies. The copy number of methanogenic 16S rDNA gene in chicken faeces ranged from log10 4Æ19 to 5Æ34 per gram wet weight when the broilers were 3, 4, 5, 9 and 12 days of age (Table 1). Methanogens in litter and house flies collected in the broiler house were log10 4Æ94 ± 0Æ10 and 5Æ51 ± 0Æ11 16S rDNA copy number per gram wet weight, respectively. Methanogenic archaea were recovered from the faecal samples by culturing in BRN medium (Balch et al. 1979; Miller and Wolin 1982) for 20 days. Methane concentrations in the headspace >100 ppm (lg ml)1) were considered positive for the presence of methanogenic bacteria in faeces. Methanogenesis was observed in 25%, 67% and 100% of the culture tubes at 5, 9 and 12 days of age, respectively (Fig. 2). Methane gas was also observed in headspace of the tubes inoculated with litter and flies (data not shown). However, cultures inoculated with dust and layer feed ration collected from the house did not produce detectable methane gas.
Days Group*
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12
ND ND 4Æ53 ± 0Æ03 ND 4Æ51 ± 0Æ02 ND 4Æ50 ± 0Æ06 ND ND ND ND ND
ND ND 5Æ03 ± 0Æ02 ND 4Æ57 ± 0Æ02 ND 4Æ62 ± 0Æ06 ND ND ND ND ND
4Æ60 4Æ63 4Æ85 4Æ97 4Æ87 4Æ92 4Æ70 4Æ82 5Æ03 4Æ37 4Æ19 4Æ27
9 ± ± ± ± ± ± ± ± ± ± ± ±
0Æ01 0Æ03 0Æ07 0Æ02 0Æ02 0Æ04 0Æ02 0Æ12 0Æ04 0Æ05 0Æ04 0Æ08
4Æ62 4Æ68 4Æ30 4Æ59 4Æ86 4Æ50 4Æ52 4Æ42 5Æ05 4Æ19 4Æ48 4Æ80
12 ± ± ± ± ± ± ± ± ± ± ± ±
0Æ05 0Æ05 0Æ04 0Æ09 0Æ11 0Æ06 0Æ04 0Æ04 0Æ18 0Æ05 0Æ14 0Æ06
5Æ34 4Æ54 4Æ61 4Æ73 4Æ72 4Æ89 4Æ80 4Æ96 4Æ81 4Æ72 4Æ86 4Æ75
± ± ± ± ± ± ± ± ± ± ± ±
0Æ14 0Æ14 0Æ05 0Æ07 0Æ06 0Æ03 0Æ06 0Æ04 0Æ01 0Æ06 0Æ11 0Æ01
* Each group was composed of 10 birds. Values are the mean ± SD of triplicate in the same RT-PCRs. ND, not determined. ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 629–634
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Methane detected (%)
100
80
60
40
20
0 5
9 Day
12
Figure 2 Per cent of culture tubes with methane concentration >100 ppm from faecal samples collected on days 5, 9 and 12. Tubes of BRN medium were inoculated with faeces and incubated at 37C under an atmosphere of 80% H2 ⁄ 20% CO2 at 200 kPa for 20 days. Methane concentrations in the tubes’ headspace were measured by gas chromatography.
Discussion Unlike methanogenic studies in humans and ruminants, the time frame of methanogen colonization in chicken caeca has not been established. Results of the current study suggest that methanogenic bacteria similar to those found in adult birds rapidly appear in the chicken faeces. We detected methane gas in cultures from 5-day faecal samples and the percentage of positive responses dramatically increased when the broilers were 9 and 12 days of age. The results from our study agreed with the earlier study (Zhu and Joerger 2003) where methanogenic bacteria were found in the caeca of very young chicks using the FISH method. In addition, it has been shown that obligate anaerobes become dominant in the chicken caecum after the first few days of life (Mead and Adams 1975). However, Marounek and Rada (1998) detected methane from in vitro incubated caecal contents only after chicks were 2 months old. The failure to detect methane gas in that study might have been because of the incubation of the cultures without headspace containing hydrogen gas, which would have more consistently supported CO2 reduction to detectable CH4 (Balch and Wolfe 1976). In addition, they incubated the chicken caecal samples for only 20 h. According to Nottingham and Hungate (1968), methane could be detected from the lowest dilution of human faecal samples after 2 days of incubation; but 20– 30 days were required for it to appear in easily measurable amounts in the highest positive dilution. 632
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An important finding in this study is that methanogenic bacteria are consistently detected in chicken faecal material prior to the time when the gastrointestinal tract is assumed to be the fully developed. The gastrointestinal tract of the chick is sterile when the bird is hatched (Conway 1997). However, bacterial colonization occurs within a few hours with the bacterial community becoming more complex as the chickens age (van der Wielen et al. 2002). According to van der Wielen et al. (2002) and Lu et al. (2003a) the microbial population found in chicken caeca was similar to those seen in the ilea in 3-day broilers. Morvan et al. (1996), and Robert and Bernalier-Donadille (2003) suggested that cellulolytic organisms might play a role in the development of a methanogenic community in the gut by providing substrates for methanogens. In humans, the presence of certain fibrolytic species (cellulolytic isolates related to Enterococcus faecalis and Ruminococcus spp.) was related to the presence of methanogens (Robert and Bernalier-Donadille 2003). A strain of Ruminococcus flavefaciens, a hydrogen-producing, cellulolytic bacterium which is known to form synthrophic associations with methanogens (Wolin et al. 1997), was isolated from the rumen of lambs 1 day after birth (Skillman et al. 2004). Enterococcus spp. and Ruminococcus spp. have also been found in caecal samples from 3-day-old chicks (Lu et al. 2003a). Ruminococcus spp. populations increased from 3 to 14 days, and after 14 days the percentage of Ruminococcous spp. was 16% of the total clones in chicken caeca (Lu et al. 2003a). In contrast, Enterococcus spp. were found to be only 1–2% of the total clones when the chicks were 3–49 days of age in caecal samples (Lu et al. 2003a). Litter and flies are potential vehicles for transmitting methanogenic archaea in birds. Bacteria in cultures of poultry litter are known to be as high as 109 CFU g)1 of material and some of the primary micro-organisms have been identified as microaerobic bacteria (Lu et al. 2003b). Faecal inoculation via the cloaca is also a conceivable exposure route as Salmonella has been shown to establish in the caeca in this manner (Corrier et al. 1994). The presence of methanogenic bacteria in litter might be because of the residual presence of these organisms from previous flocks. The presence of methanogens in fly samples might be due either to methanogen contamination from chick faeces or methanogen colonization in this insect. House flies carry heterogeneous mixtures of organisms and have been considered to be a source of Campylobacter colonization in broiler chickens (Newell and Fearnley 2003; Ekdahl et al. 2005; Nichols 2005). Methanogens, however, may also be part of normal microbial population in the house fly. Methanobrevibacter spp. have been isolated from the cockroach hindgut (Gijzen et al. 1991) and the hindgut content of termite
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