Effect of Anaerobic Digestion on Oocysts of the Protozoan

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1988, p /88/ $02.00/0 Copyright 1988, American Society for Microbiology Vol. 54, No. 10 Effect of Anaerobic Digestion on Oocysts of the Protozoan Eimeria tenellat MIAU-RONG LEE AND JASON C. H. SHIH* Biotechnology Laboratory, Department of Poultry Science, North Carolina State University, Raleigh, North Carolina Received 21 March 1988/Accepted 8 July 1988 The effect of anaerobic digestion of poultry waste on oocysts of the protozoan Eimeria tenella, a common enteric pathogen that causes coccidiosis in poultry, was investigated in this study. Thermophilic (50 C) and mesophilic (35 C) anaerobic digestors, with poultry manure as the substrate, were inoculated with the oocysts. The oocysts were damaged during anaerobic digestion, as determined by morphological change and loss of their ability to sporulate. The recovered oocysts were tested for their infectivity in young chicks, as measured by body weight gain, mortality, and cecal lesions. Oocysts lost all their infectivity during thermophilic digestion, while oocysts subjected to mesophilic digestion remained moderately infective in comparison with untreated oocysts, which produced severe coccidiosis, high mortality, and low body weight gain in chicks. Oocysts were inactivated at 50 C when they were suspended in digestor fluid or saline. Inactivation at 35 C was significantly stronger in the digestor fluid than in the saline, which implied that factors other than temperature were involved in the lethal effect of anaerobic digestion on protozoan oocysts. In this study we demonstrated that the treatment of animal waste by anaerobic digestion, especially at a thermophilic temperature, has the benefits of pathogen control and protection of human and animal health in a farm environment. Many attempts to develop methods for economical treatment and utilization of poultry waste have been made (26). One of the methods is anaerobic digestion, which is made up of a series of microbial actions which convert the organic content of the waste to biogas, a mixture of CH4 and C02, in the absence of exogenous electron acceptors such as oxygen (13, 14). A simple and low-cost poultry waste digestor system has been developed in our laboratory for operation and poultry farms (31). The biogas produced can be used as a source of energy (17a). The liquid effluent, which contains many nutrients, can be used for irrigation or aquaculture (26). The solid residue separated from the effluent can be used as fertilizer or as an animal feed supplement (22, 30). When all products are fully utilized, anaerobic digestion can be an attractive and economical option for waste treatment on an animal farm (27). Commonly, untreated poultry waste contains pathogenic microflora. Anaerobic digestion can destroy pathogenic microorganisms and reduce disease arising from fecal contaminations. Animal pathogens, such as Salmonella sp., enteroviruses, and Ascaris sp., are reduced in the anaerobic digestion of municipal sewage (5). Thermophilic digestion enhances the destruction of pathogenic organisms (4). However, little work has been done to investigate pathogen control in anaerobic digestion of farm animal waste. Ninety percent reduction of salmonellae in anaerobic digestors of pig wastes for 4 days has been reported (6). Low numbers of Salmonella sp., persisted in the digestor even after 70 days, however. Recently, complete destruction of salmonellae and fecal coliforms in a thermophilic poultry waste digestor was discovered in our laboratory (25, 26). Some phytopathogenic microorganisms were found to be reduced in a hog waste digestor (33). Coccidiosis caused by the protozoan Eimeria spp. is one * Corresponding author. t This is paper number of the Joumal Series of the North Carolina Agricultural Research Service, Raleigh. of the most prevalent and economically devastating diseases of poultry. An important factor in the epidemiology of coccidiosis is the survival of oocysts that are shed in the excreta of infected hosts, which is the primary source of infection. Chickens may continue to shed oocysts even after they have recovered from the acute effects of the disease (23). A waste treatment process which destroys oocysts interrupts the life cycle of coccidia and prevents a disease outbreak (9). This study was carried out to determine the survival and infectivity of sporulated and unsporulated oocysts of Eimeria tenella in thermophilic and mesophilic anaerobic digestion. MATERIALS AND METHODS Anaerobic digestor. The operation of laboratory anaerobic digestors has been described previously (16, 27a). One-liter glass digestors were used in this study. The digestors were started with microflora that are indigenous to the manure of laying hens. The mesophilic (35 C) digestors had a retention time of 10 days, while the thermophilic (50 C) digestors had a retention time of 5 days. Analysis of the total solid, volatile solid, total nitrogen, and ammonia nitrogen contents of the layer manure, digestor influent, and digestor effluent was conducted by standard methods (1). Biogas composition was determined by gas chromatography (16). Oocysts. Oocysts of E. tenella were obtained from Donald J. Nelson (Burroughs Wellcome Co., Research Triangle Park, N.C.). Methods for handling the parasites, such as isolation, sporulation, and counting of oocysts, were the same as those described previously (21, 24). Fresh, unsporulated oocysts for each trial were isolated from cecal contents of 3-week-old chicks, 7 days after they were infected with E. tenella oocysts. Isolation of oocysts. Cecal contents or digestor effluent containing oocysts were diluted with deionized water and mixed thoroughly. The mixture was filtered sequentially through 40- and 100-mesh sieves and centrifuged at 1,800 x g for 15 min, and the supernatant was discarded. After two 2335

2 2336 LEE AND SHIH FIG. 1. Photomicrographs of sporulated (upper) and unsporulated (lower) E. tenella oocysts. Magnification, x 3,100. washings, the sediment was suspended in a saturated NaCl solution and centrifuged at 126 x g for 10 min. The oocystcontaining scum was removed with a Pasteur pipette from the top of the salt solution. Three crops of oocysts were obtained the same way by repeated flotations. The combined crops of oocysts, which were 5 times diluted with water, were centrifuged at 500 x g for 15 min to obtain an oocyst precipitate, which was washed three times in water by centrifugation before it was counted in a McMaster chamber under a microscope (21). The recovery of oocysts through this procedure was about 50%. Sporulation of oocysts. The fresh or digestor-treated unsporulated oocysts were suspended in a shallow layer (5 mm deep) of 2.5% potassium dichromate solution in a 50-ml flask and incubated at 30 C with constant shaking for 3 days, to allow the oocysts to sporulate. The sporulated oocysts had four sporocysts which were easily detected under the microscope (Fig. 1). Anaerobic digestion treatment. Unsporulated or sporulated oocysts were added to the digestors, which were fed a manure slurry daily. The digestors were mixed well to ensure homogeneous distributions of oocysts in the digestors. Oocyst numbers were counted in each daily effluent sample. As a comparison, the unsporulated or sporulated oocysts were put in saline (0.85% NaCl) and incubated at 35 or 50 C. All treated oocysts were then recovered to determine their ability to sporulate (for unsporulated oocysts) and to infect chicks. Oocysts in each treatment group were observed by light microscopy so that their morphological variation could be examined. Hungate test tubes. The results obtained from the digestor experiments were confirmed by the anaerobic Hungate tube technique (3). Hungate tubes containing 107 to 108 sporulated or unsporulated oocysts were incubated with digestor fluid that was withdrawn from the thermophilic or mesophilic digestor or saline for 24 h at 35 or 50 C. The tubes were then emptied to collect oocysts for the infectivity test. Experimental animals. One-day-old male broiler-type chicks (Ross Arbor Acre strain) were reared in a wirefloored heated Petersime chick battery in an air-conditioned (21 C) room with a 16-h light and 8-h dark cycle. Feed that was free of anticoccidial drugs and water were available ad libitum. The chicks were raised to 15 days of age and were then used for the infectivity test. APPL. ENVIRON. MICROBIOL. Infectivity test. Fifteen-day-old coccidia-free chickens were weighed and moved into an isolation room, with room temperature kept at 25 C. The isolation room and equipment were thoroughly cleaned and disinfected before the chicks were placed inside. Each bird was in an individual suspended wire cage with individual feeding and watering facilities. All the treatments were tested independently in groups of five birds. Before inoculation, 2.5% potassium dichromate, in which oocysts were stored, was replaced by saline. The number of oocysts in each group were counted with a hemacytometer. Approximately 105 to 106 oocysts were intubated into the crop of each chick with a 1-ml sterile plastic pipette. For uninfected controls, chicks received 1 ml of saline. For the infected controls, chicks received a dose of fresh, untreated, and sporulated oocysts. The infectivity of treated oocysts was assessed by mortality, cecal lesions, reduced body weight gain, and the presence of oocysts in cecal scrapings. Seven days after infection, all the surviving birds were weighed and sacrificed by cervical dislocation. Cecae were removed immediately. All of the birds that died prior to this time were necropsied and examined macroscopically and microscopically to confirm the existence of coccidiosis. The cecal lesions induced by E. tenella were scored by the technique of Johnson and Reid (18) on a scale of 0 to 4: 0, no lesions; 1, a few scattered petechiae seen on the surface of the mucosa, with little change in color of the cecal walls or contents, the normal cecal contents present; 2, presence of more blood and lesions and a slight thickening of the cecal TABLE 1. Performance of the laboratory thermophilic and mesophilic anaerobic digestorsa Parameter Thermophilic digestor Mesophilic digestor Temp, C Influentb TS (%) 7.5 ± 0.4 (6)C 7.5 ± 0.4 (6) VS (%) 5.9 ± 0.3 (6) 5.9 ± 0.3 (6) TN (mg/liter) (6) 6076 ± 48 (6) NH3-N (mg/liter) 861 ± 120 (6) 861 ± 120 (6) RT (days) 5 10 Loading (g of VS/liter 11.7 (6) 5.9 (6) per day) ph Effluentb TS (%) 5.5 ± 0.2 (5) 5.3 ± 0.2 (5) VS (%) 3.0 ± 1.0 (5) 2.8 ± 0.2 (5) TN (mg/liter) NDd 6122 ± 243 (6) NH3-N (mg/liter) 4,091 ± 165 (8) 4525 ± 248 (6) VS reduction (%) 48.5 ± 1.9 (5) 53.5 ± 5.2 (5) ph 7.1 to to 8.0 Gas rate (liters/liter per day) Biogas 3.0 ± 0.1 (7) 2.5 ± 0.1 (6) CH4 1.5 ± 0.1 (7) 1.5 ± 0.1 (6) % CH ± 3.0 (7) 60.7 ± 2.2 (6) Gas yield (liters/kg of VS) Biogas 257 ± 11 (7) 420 ± 15 (6) CH4 128 ± 7 (7) 255 ± 12 (6) a Values are means ± standard errors of the mean. b Abbreviations: TS, total solids; VS, volatile solids; TN, total N; RT, retention time. ' Numbers in parentheses are the number of replicates. d ND, Not determined.

3 VoL. 54, 1988 ANAEROBIC DIGESTION EFFECT ON COCCIDIA 2337 RESULTS FIG. 2. Recovery of E. tenella oocysts from thermophilic and mesophilic digestors (n = 5). RT, Retention time. wall; 3, large amounts of blood or cecal cores present, cecal walls greatly thickened, and absence of normal fecal contents in the ceca; 4, very severe coccidial infection characterized by cecal swelling and bleeding. Dead birds were scored as 4. Statistics. Analysis of variance and the Duncan multiple range test (28) were used to analyze the pooled lesion score and body weight gain of birds in all three trials of the infectiviiy studies. TABLE 2. Anaerobic digestion. The performance of the digestors was monitored before and after oocysts were added for a period of 6 days in each experiment. A summary of the characteristics of the digestor influent (manure slurry), effluent, gas rate, and gas yield is presented in Table 1. Enumeration of oocysts in digestors. A single dose of unsporulated oocysts was added to each of the digestors. Average relative counts, i.e., the daily observed oocyst count over the oocyst counts of day 0 for a period of 5 days after the initial inoculations, are shown in Fig. 2. Theoretical count curves obtained from the retention times or dilution factors of both digestors are also shown in Fig. 2. The differences between the observed curve and theoretical dilution curve were not significant in either digestor. Sporulation of treated oocysts. A definite decrease in sporulation of oocysts treated in the digestor for 1 day was observed (Table 2). The sporulation rate of freshly isolated unsporulated oocysts was 89 to 94%. However, none of the oocysts sporulated after the 1-day treatment in the thermophilic digestor and with saline at 50 C. The mesophilic digestor-treated oocysts had a sporulation range from 0.2 to 8.5%. After treatment with saline at 350C, a few oocysts sporulated in 1 day, but 86 to 92% of these oocysts treated with saline at 35 C sporulated in 3 days. Thus, oocysts Sporulation and infectivity of unsporulated oocysts after treatments Dose Body wt gain Mortality Presence of Treatment Expt Sporulation (%) (oocysts, 105)/bird (g [%])' (no. dead/total no.) oocystsb Lesion score' Th,ermophilic Digestor (95) 0/5 5/5 0.5 t t 31 (118) 0/4 0/ t 62 (137) 0/4 1/4 0.1 t t 76 (132) 0/5 2/5 0.2 t 0.3 Saline t 40 (96) 0/5 NDd t 83 (61) 0/4 0/ t 39 (109) 0/5 1/5 0.1 t t 56 (169) 0/5 2/5 0.3 t 0.5 Mesophilic Digestor t 48 (26) 1/7 7/7 2.1 t t 33 (751 0/4 4/4 1.0 t t 98 (85) 0/3 3/3 1.0 ± t 32 (135) 0/5 5/5 0.8 t 0.5 Saline t 77 (52) 1/6 ND 2.6 ± ± 23 (41) 0/4 ND 3.5 t ± 25 (47) 2/5 5/5 2.7 ± ± 28 (36) 2/5 5/5 2.8 ± 1.2 Control Infection ±18 (42) 2/5 ND 3.7 ± (12) 3/7 6/7 3.3 ± ± 67 (48) 1/6 6/6 2.8 ± ± 20 (75) 2/6 6/6 3.0 ± 1.0 Noninfection ± 72 (100) 0/5 ND ±45 (100) 0/4 0/ (100) 0/5 0/ (100) 0/6 0/6 0 a Percentage of weight gain of infected birds/weight gain of noninfected controls in the same trial. b Birds positive for oocysts in ceca/total number of birds. Lesion score from 0 to 4. d ND, Not determined.

4 2338 LEE AND SHIH APPL. ENVIRON. MICROBIOL. Downloaded from FIG. 3. E. tenella oocysts after treatment in the thermophilic digestor (A) and after treatment in the mesophilic digestor and then sporulation in 2.5% K2Cr2O7 for 3 days at 30 C (B). Magnification, x3,400. treated with saline at 35 C for 1 day still retained their potential ability to sporulate. Morphology of treated oocysts. The photomicrographs of unsporulated oocysts treated for 1 day in digestors and with saline are shown in Fig. 3. The thermophilic digestor-treated oocysts retained their outer shape, but their cytoplasm was greatly condensed or reduced (Fig. 3A). None of the thermophilic digestor or oocysts treated with saline at 50 C sporulated. Most of the mesophilic digestor-treated oocysts also had a condensed cytoplasm, with some oocysts exhibiting aberrant sporocysts (Fig. 3B). A few oocysts completely sporulated and contained four sporocysts, each with two sporozoites. Most of the oocysts treated with saline at 35 C completely sporulated as normal. For sporulated oocysts, no morphological changes were detected after any of these treatments. Infectivity of treated oocysts. Table 2 also shows the in vivo infectivity of unsporulated oocysts treated in a digestor or with saline. The infected control groups of chicks exhibited extensive cecal lesions and reduced weight gain among the survivors. The mortality of chicks in the infected control groups was 17 to 43%. Chicks in the uninfected control groups had no lesions and no oocysts were found in the ceca. Chicks inoculated with oocysts treated in a thermophilic digestor or with saline at 50 C had very low lesion scores and exhibited normal or higher body weight gains. The oocysts treated in mesophilic digestors caused moderate cecal lesions and a 25% reduction in body weight gains. Chickens inoculated with oocysts treated with saline at 35 C had high lesion scores and 60% weight loss. The severity of infection in oocysts treated with saline at 35 C and infected control groups was similar. Table 3 shows the in vitro infectivity of sporulated oocysts treated in a thermophilic digestor and with saline. Chicks infected with oocysts treated with saline at 50'C had mild lesion scores and normal body weight gain in one trial. In the on September 15, 2018 by guest

5 VOL. 54, 1988 ANAEROBIC DIGESTION EFFECT ON COCCIDIA 2339 TABLE 3. Infectivity of sporulated oocysts after the thermophilic (50 C) treatment Dose Body wt gain Mortality Presence of Treatment Expt (oocysts, 105)/bird (g [%])a (no. dead/total no.) oocystsb Leson score Digestor ± 64 0/5 0/ ± 65 0/4 0/4 0.3 ± ± 60 (115) 0/5 4/5 0.5 ± 0.4 Saline ± 34 0/5 5/5 1.2 ± ± 15 0/4 4/5 1.6 ± ± 28 (167) 0/5 0/5 0 Control Infection ± 20 (75) 2/6 6/6 3.0 t 1.0 Noninfection ± 51(100) 0/6 0/6 0 a Percentage of weight gain of infected birds/weight gain of noninfected controls in the same trial. b Birds positive for oocysts in ceca/total number of birds. Lesion score from 0 to 4. third experiment, however, there were no lesions, and no This might have been due to the extremely strong and unique oocysts present in the cecal smears were observed. Chicks oocyst wall that kept the oocyst shape intact. The variation infected with oocysts that were treated in the thermophilic in counts was high because of sampling variation from the digestor had no infectivity observed in the first experiment. digestors. The killing effect of the thermophilic digestor on In the third experinment, however, a very mild lesion and E. tenella oocysts was better than that of the mesophilic oocysts were detected in ceca. digestor (Table 5). Most of these digestor-treated oocysts A Hungate tube trial was performed to confirm the results lost their ability to sporulate and to infect chicks. Fitzgerald of digestor treatnments. The conditions of these anaerobic and Ashley (11) have also reported that rabbit coccidium tubes were similar to those of laboratory digestors. The Eimeria stiedai oocysts, which were recovered from the results of in vivo infectivity (Table 4) confirmed the obser- sample of a 38 C anaerobic digestor, were all unable to vations made in digestors. Treatments at 50 C completely sporulate. inactivated the oocysts, but treatments at 35 C only partially Many factors, such as temperature, absence of oxygen, inactivated the infectivity of the oocysts. volatile fatty acids, ammonia, and antibioticlike substances, Analysis of variance and the Duncan multiple range test in anaerobic digestors might be lethal to the coccidial were used to analyze pooled lesion score and body weight oocysts. Temperature has been recognized to be one of the gain data from all infectivity study experiments. The com- critical factors in affecting the viability of oocysts (19). In bined results in Table 5 show that thermophiic treatments this study, temperature was the major factor which affected were more effective than mesophilic treatments in destroy- oocyst viability in anaerobic digestors. No sporulation was ing both sporulated and unsporulated oocysts. detected after unsporulated oocysts were treated with saline at 50 C for day. Unsporulated oocysts treated with saline DISCUSSION at 35 C for 1 day, however, still exhibited 90% sporulation There was no significant reduction in the number of and caused severe infections of chickens. Sporulated oocysts during anaerobic digestion in both digestors (Fig. 2). oocysts treated in a digestor or with saline at 50 C were TABLE 4. Infectivity of oocysts treated in HIungate tubes Oocysts (treatment Dose Body wt gain Mortality Presence of Lesion scorec temp) (oocysts, 105)/bird (g [%])a (no. dead/total no.) oocystsb Unsporulated (50 C) Digestor fluid ± 60 (152) 0/5 1/5 0.1 ± 0.2 Saline ± 31 (128) 0/5 1/5 0.1 ± 0.2 Unsporulated (35 C) Digestor fluid ± 31 (117) 1/6 6/6 1.8 ± 1.2 Sporulated (50 C) Digestor fluid ± 64 (140) 0/4 2/4 0.3 ± 0.3 Saline ± 46 (145) 0/5 1/5 0.1 ± 0.2 Sporulated (35 C) Digestor fluid ± 11 (71) 1/5 5/5 2.1 ± 1.4 Control Infection (45) 3/4 4/4 4.0 Noninfection ± 70 (100) 0/5 0/5 0 a Percentage of weight gain of infected birds/weight gain of noninfected controls. b Birds positive for oocysts in ceca/total number of birds. c Lesion score from 0 to 4.

6 2340 LEE AND SHIH TABLE 5. Summary of in vivo infectivity of treated oocysts in all experiments Oocysts No. of Average body wt Average (treatment temp) expt gain (g) of survivors lesion score Unsporulated (50 C) Digestor a.0.18b.c Saline a,d 0.10' Unsporulated (35 C) Digestor e Saline b 2 god Sporulated (50 C) Digestor a,d 0.28bo' Saline a 0.73b Sporulated (35 C) Digestor be 2. lod Infected control Noninfection control ad 0.00 avalues within a column followed by different letters are significantly different (P < 0.05). APPL. ENVIRON. MICROBIOL. dead, as determined by their lack of infectivity in chicks. Our observations are consistent with those made in other studies (7, 10), in which the inactivation of oocysts of E. tenella was demonstrated at elevated temperatures. The thermophilic digestor-treated unsporulated oocyst and sporulated oocyst groups did not have significantly different infectivities, although the average lesion score of the thermophilic digestor-treated unsporulated group was somewhat higher than that of the sporulated group (Table 5). It has been suggested that the unsporulated oocysts are more susceptible to the physical and chemical agents than the sporulated oocysts are (9). Unsporulated oocysts may be more metabolically active and sensitive than sporulated oocysts, which are in a resting stage. However, based on results of the present study, it seems that the 50 C anaerobic digestor has similar and strong killing effects against both sporulated and unsporulated oocysts. Results for the mesophilic digestor and saline groups were significantly different in the infectivity studies (Tables 2 and 5). The mesophilic digestor killed about 90 to 99% of the unsporulated oocysts, but 35 C saline had little effect. This difference must have been due to factors other than temperature. In both digestors, the ammonia nitrogen concentration was high (about 4,000 to 4,500 mg/liter). Ammonia, one of the small molecules that can penetrate the wall of the oocyst, is lethal to coccidia oocysts (15, 19). A 1% solution of ammonia is known to kill 100% of the oocysts of E. tenella in 24 h (19). Thus, the high ammonia level in these digestors could have affected the viability of the oocysts. Anaerobic conditions have also been noted to be unfavorable for oocyst sporulation and viability (i9). The effect of volatile fatty acids in the digestor might not significantly influence oocyst survival. Farquhar et al. (8) studied the prevention of sporulation of bovine coccidia Eimeria bovis by ensiling a manure-blended diet. They found that oocyst survival declined with higher ensiling temperatures and prolonged incubation. Their analysis revealed that acetate and propionate production was inversely correlated to oocyst survival, and their influence was considered minimal. In an earlier study, it was found that 30% of Eimeria magna oocysts still sporulated after 72 h in 5 M acetic acid (19). During this study, the mesophilic digestor was operated in good condition, with parameters and gas rate comparable to those in other studies (2, 13). However, the performance of the thermophilic digestor did not seem as good as that reported previously (16, 31) and as operated in our laboratory. The commonly observed biogas volumetric rate has been 4.0 to 4.5 liters/liter per day and the biogas yield has been 450 to 500 liter/kg of volatile solids with 55 to 65% methane (16, 31). The poor performance of this thermophilic digestor was believed to be caused by the lower ph value (7.1 to 7.2) than those used in previous studies and in this one (ph 7.5 to 8.0). The toxicity of volatile fatty acids to methanogenesis (17, 29) or other effects (20) may be involved. However, the digestor was considered to be acceptable for pathogen inactivation in this study. The ammonia nitrogen concentrations were 4,091 and 4,525 mg/liter in thermophilic and mesophilic digestors, respectively. The toxicity of an ammonia nitrogen concentration of greater than 1,500 mg/liter to methanogenesis was not observed in this study. In some other studies digestion was also satisfactory at elevated ammonia nitrogen concentrations (12, 20, 32, 34). The successful digestion was attributed to acclimation or selection of the methanogenic bacteria to high levels of ammonia or cation. In the study of anaerobic digestion of poultry manure, Hills and Ravishanker (13) found that the methanogens appeared to acclimate successfully to high concentrations of ammonia nitrogen (4.5 g/liter). 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