Prolonged Survival of Campylobacter Species in Bovine Manure Compost

The persistence of naturally occurring campylobacteria in aerobic compost constructed of manure from beef cattle that were administered chlortetracycline and sulfamethazine (AS700) or from cattle not administered antibiotics (control) was examined. Although there were no differences in population sizes of heterotrophic bacteria, the temperature of AS700 compost was more variable and did not become as high as that of control compost. There were significant differences in water content, total carbon (C), total nitrogen (N), and electrical conductivity but not in the C/N ratio or pH between the two compost treatments. Campylobacteria were readily isolated from pen manure, for up to day 15 from control compost, and throughout the active phase of AS700 compost. Campylobacter DNA (including Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, and Campylobacter jejuni) was detected over the ca. 10-month composting period, and no reductions in quantities of C. jejuni DNA were observed over the duration of the active phase. The utilization of centrifugation in combination with ethidium monoazide (EMA) significantly reduced (>90%) the amplification of C. jejuni DNA that did not originate from cells with intact cell membranes. No differences were observed in the frequency of Campylobacter DNA detection between EMA- and non-EMA-treated samples, suggesting that Campylobacter DNA amplified from compost was extracted from cells with intact cell membranes (i.e., from viable cells). The findings of this study indicate that campylobacteria excreted in cattle feces persist for long periods in compost and call into question the common belief that these bacteria do not persist in manure.Campylobacter jejuni and, to a lesser extent, Campylobacter coli incite serious acute and chronic afflictions. Enteritis caused by C. jejuni (i.e., campylobacteriosis) is the most common cause of bacterial enteritis in Canada (http://dsol-smed.phac-aspc.gc.ca/dsol-smed/ndis/index-eng.php). Although the epidemiology of campylobacteriosis is poorly understood, sporadic outbreaks of campylobacteriosis involving contaminated water have occurred when water treatment has failed. The most serious outbreak in Canada occurred in Walkerton Ontario in 2000; more than 2,300 people became infected with waterborne Escherichia coli O157:H7 and/or C. jejuni originating from cattle feces (3). Alberta, Canada, possesses a very large beef cattle population (≈6 million animals) primarily concentrated in the southern region of the province, and ≈2 million of these animals are in finishing feedlots (1). Large quantities of manure are produced by feedlot cattle. For example, in the Chinook Health Region of Southwestern Alberta in which Lethbridge is situated, there are ≈700,000 cattle in feedlots at any given time, producing ≈12 million kg of manure (fresh weight) per day. Several Campylobacter species, including C. jejuni and C. coli, are frequently shed in beef cattle feces in large numbers (15, 16). Although the impact of cattle-borne campylobacters on human health has not been definitely determined, the southern region of Alberta possesses one of the highest rates of campylobacteriosis in Canada among its human inhabitants, concomitant with the very high density of cattle in this region.Large-scale windrow composting of bovine manure from intensive cattle operations is practiced by some Alberta feedlots. Composting is an aerobic process in which organic matter in manure is stabilized into a humus-like product (30). The process results in water loss and mass reduction, nutrient transformation (22), alteration of physical structure (23), elimination of weed seeds (21), and the inactivation of coliform bacteria (25), protozoan cysts and oocysts (34), and viruses (39). Limited research has investigated the impact of manure management systems, such as aerobic composting, on deactivation of campylobacters. Furthermore, the impact of antimicrobial agents excreted into the manure on the efficacy of the composting process on Campylobacter deactivation has not been investigated. Most studies conducted to date have indicated that campylobacters do not persist well in solid manure once excreted (7, 11, 12, 26, 32, 39). Although it is difficult to isolate or enumerate Campylobacter species within microbiologically complex substrates, molecular detection and/or quantification methods have not been extensively applied to study the persistence of campylobacteria. Furthermore, the persistence of naturally shed campylobacteria has largely been overlooked. Thus, the overall objective of the current study was to measure the ability of campylobacteria naturally shed in bovine feces to persist in manure compost using a combination of culture- and culture-independent methods. Specific objectives were (i) to develop and utilize a centrifugation method to facilitate isolation and detection of DNA from Campylobacter species in bovine manure compost, (ii) to apply qualitative and quantitative PCR methods to evaluate persistence of campylobacteria in compost, (iii) to validate the molecular methods used to amplify DNA from viable cells, and (iv) to contrast the persistence of Campylobacter species in composted manure obtained from beef cattle maintained on a diet supplemented with chlortetracycline and sulfamethazine (AS700) with composted manure from animals not administered antimicrobial agents.