Published online 1 March 2006
Published in J Environ Qual 35:680-687 (2006)
DOI: 10.2134/jeq2005.0273
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Waste Management
Survival of Cryptosporidium parvum Oocysts in Calf Housing Facilities in the New York City Watersheds
A. S. Collicka,
E. A. Fogartyb,
P. E. Zieglerc,
M. T. Waltera,
D. D. Bowmanb and
T. S. Steenhuisa,*
a Department of Biological & Environmental Engineering, Cornell University, Ithaca, NY 14853
b Department of Microbiology & Immunology, Cornell University, Ithaca, NY 14853
c Department of Population Medicine & Diagnostic Science, Cornell University, Ithaca, NY 14853
* Corresponding author (tss1{at}cornell.edu)
Received for publication July 14, 2005.
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ABSTRACT
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Pathogen contamination of the public drinking water supply in the New York City watersheds is a serious concern. New York City's Watershed Agriculture Program is working with dairy farms in the watersheds to implement management practices that will reduce the risk of pathogens contaminating the water supply. Solar calf housing (SCH) was suggested as a best management practice (BMP) to control Cryptosporidium parvum, a common protozoan parasite that causes disease in humans. This BMP targets young calves because they are the primary source of C. parvum in dairy herds. The objective of this project was to assess and compare the survivability of C. parvum in SCH and in conventional calf housing (CCH), usually located in the main barn. C. parvum oocysts were secured in sentinel chambers and placed in SCH and CCH bedding on four farms. The chambers were in thermal, chemical, and moisture equilibrium with their microenvironments. An oocyst-filled control chamber, sealed from its surroundings, was placed near each chamber. Chambers and controls were sampled after 4, 6, and 8 wk. Oocyst viability in the chambers decreased to less than 10% in warm months and between 15 and 30% in the winter months. The viability of the control oocysts was similar to the chambers during warm months and generally higher during winter months. There was no significant (P > 0.05) difference in the viability decrease between SCH and CCH. Although oocyst viability was similar in both types of calf housing, SCH allow contaminated calf manure to be isolated from the main barn manure and potentially managed differently and in a way to decrease the number of viable oocysts entering the environment during field spreading.
Abbreviations: BMP, best management practice • CCH, conventional calf housing • FITC, fluorescein isothiocyanate • PI, propidium iodide • SCH, solar calf housing
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INTRODUCTION
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THE ZOONOTIC POTENTIAL of Cryptosporidium parvum is a serious concern in watersheds supplying public drinking water because it can infect and cause disease in humans. In watersheds where agriculture prevails, such as in the New York City watersheds, livestock manure, spread on fields as fertilizer, is a potential source of C. parvum oocysts that may contaminate drinking water. The infectious stage of C. parvum, the oocyst, is quite resistant and has been found to survive several weeks to months in soil (Jenkins et al., 1999; Kato et al., 2001b) and water (Medema et al., 1997). Agricultural cropping and grazing land are suspected source areas of this protozoan parasite for several documented, waterborne outbreaks of cryptosporidiosis (Smith, 1998; Fricker and Crabb, 1998; Solo-Gabriele and Neumeister, 1996; Richardson et al., 1991; Fayer et al., 2000). Although solid epidemiological evidence does not exist to fully link these outbreaks to agricultural sources, the management of oocyst contribution from livestock warrants investigation.
Most of the water from the New York City watersheds (>90%) remains unfiltered. By complying with 1997 and 2002 Filtration Avoidance Determinations from the USEPA, the Delaware and Catskill Watersheds (Fig. 1
) may remain unfiltered until specified criteria can no longer be met. The criteria specified in the Surface Water Treatment Rule of 1989, issued through the Safe Drinking Water Act of 1974, require the maintenance of healthy levels of several source water quality measures, including turbidity, fecal coliform, and disinfection specifications for unfiltered systems. The Surface Water Treatment Rule also mandated the establishment of watershed control programs, including programs to minimize pathogen contamination (New York City Department of Environmental Protection, 2001). Currently, disinfection requirements are satisfied by the addition of chlorine to the water in the distribution system. The Watershed Agricultural Program (WAP) and its operational and administrative organization, the Watershed Agricultural Council (WAC), are part of the New York City watershed control program that works with the agricultural community to implement farm-based practices that protect water quality by reducing and preventing pollution (New York City Department of Environmental Protection, 2001).
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Fig. 1. The major basins of the Delaware and Catskill Watersheds located in southeastern New York. The four participating farms are located within the Cannonsville Basin.
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Several of these farm-based practices, or best management practices (BMPs), have been implemented on farms within the New York City watersheds to reduce nutrient loading, chemical pollution, and pathogen contamination. The installation of solar calf housing (SCH) on dairy farms is one of the BMPs being implemented to manage C. parvum in the New York City watersheds. Calves are targeted in C. parvum management because a genetic isolate of C. parvum (Genotype 2) shed by calves can cause infection in humans (Peng et al., 1997; Fayer et al., 2000). Calves are also the population of cattle most at risk of infection and they can shed a large quantity of oocysts (up to 106 to 107 oocysts per gram of feces) during serious infections (Blewett, 1989; Angus, 1990). According to Mohammed et al. (1999), various management techniques within calf housing facilities are associated with either the increase or decrease of infection with C. parvum in calves. For example, the use of ventilation, daily disposal of soiled bedding, and the daily addition of bedding to preweaned calf areas are associated with decreased infection risk. The objective of SCH (also referred to as greenhouse calf barns) is to provide cleaner, better ventilated, more accessible, and, ultimately, healthier conditions for calf raising (Kammel et al., 1996). During a survey of 10 SCHs in Delaware County, New York, positive effects on the health and, in particular, the weight gain, of calves raised in these facilities were reported in comparison to those calves raised in conventional calf housing (CCH) (Huneke and Hilson, 1999).
In many cases, SCH replaced CCH, which involved raising calves in the main barn adjacent to the lactating cows. Solar and conventional calf housing often have different flooring, bedding, lighting, and ventilation (Fig. 2
). However, the most distinct management difference between these two housing facilities is the extended retention time of calf bedding in the SCH compared to the daily to near weekly removal of calf bedding in the CCH. Eventually, the soiled bedding from both facilities is spread on fields.
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Fig. 2. Generalized differences in management techniques employed in solar and conventional calf housing facilities.
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Three conditions are necessary for C. parvum to contaminate a public water supply: (i) oocysts must be present in contaminated wastes within the water supply's hydrologic system, (ii) there must be sufficient oocyst survivability in the environment, and (iii) there must be mechanisms to transport the oocysts to the water supply (e.g., hydrological runoff events of sufficient energy and duration) (Walker et al., 1998). Therefore, managing potentially contaminated animal waste involves a series of approaches addressing each of the above conditions. Specifically, management includes controlling the magnitude of the potential pathogen source through herd health initiatives, isolating and effectively treating collected animal waste, and exposing pathogens to environmental stresses during waste storage (Walker et al., 1998). Solar calf housing conceptually facilitates these approaches, but the effectiveness has not been tested. This study investigates the survivability of C. parvum oocysts in SCH and CCH. Oocyst management can be considered effective if oocysts are inactivated before being released into the environment because only viable oocysts are capable of causing infection in a host. This study determined the survival of oocysts in SCH and CCH using sentinel chambers (Jenkins et al., 1999, 2002; Kato et al., 2001b, 2002). Previous field studies investigated oocyst survival using these chambers in compost piles (Jenkins et al., 1999), in manure-amended soil (Jenkins et al., 1999), and in field soil (Kato et al., 2001b), but little is known on the survival of oocysts in calf housing facilities where the oocysts are initially shed. Developed by Jenkins et al. (1999), the chambers were designed to secure oocysts inoculated in a chamber medium (usually soil or mixed animal waste solids) and to allow for the oocysts to equilibrate to external conditions through permeable mesh. After retrieval, oocysts were extracted and their viability determined by a dye permeability assay (Campbell et al., 1992; Robertson et al., 1992; Jenkins et al., 1997; Kato et al., 2001a). Although other assays, such as infectivity and excystation assays, are available to determine infectivity of oocysts, the excystation of oocysts during in vitro infectivity and excystation assays may not be comparable to the actual excystation that occurs in the host (Kato et al., 2001a). Mouse infectivity assays are prohibitively expensive. Therefore, to obtain the percentage of oocysts, which are truly dead (inactivated), the dye permeability assay was deemed most appropriate and most consistent to utilize for this study.
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MATERIALS AND METHODS
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Calf Housing Facilities
Four dairy farms located in the Cannonsville Basin of the Delaware Watershed System in Delaware County, New York (Fig. 1) participated in this study. During an initial visit to each of these farms, direct fecal samples were collected from all calves under the age of 30 d and at least one sample from each of the farms was positive for C. parvum oocysts as determined by the standard, centrifugation concentration sugar (specific gravity = 1.33) flotation technique and bright field and phase contrast microscopy (Wade et al., 2000). Calves on two of the farms were raised in individual pens in SCHs, constructed within 5 yr of this study. Calves on the remaining two farms were conventionally raised in the dairy barn with the rest of the dairy herd, leashed to the barn wall or to a tie rail, or secured in individual or group stalls.
Sentinel Chamber
Sentinel chambers were used to determine the survival of C. parvum oocysts exposed to the calf bedding in SCH and CCH. The chambers were fully assembled (Excelsior Sentinel, Newfield, NY) and consisted of a 2.5-cm-long, 1.3-cm-diameter polycarbonate tube enclosed on both ends by 10-µm nylon mesh. The mesh was secured with open-ended polycarbonate caps (Fig. 3
). Since the adherence of soil particles and oocysts increased the overall size of an oocyst beyond the diameter of a mesh opening, the mesh securely contained the oocysts within the chamber and allowed for maximum exposure and environmental equilibration (Jenkins et al., 1999).
Chamber Preparation
Soiled calf bedding from the four farms was collected, dried at 55°C for >24 h, ground, sieved (2-mm diameter openings), and stored at 4°C for use in the chambers. Oocysts were collected from the feces of naturally infected 7- to 14-d-old calves on dairy farms in Tompkins County, New York, and purified according to a sucrose-Percoll (Pharmacia, Uppsala, Sweden) flotation method (Jenkins et al., 1997). Chambers were designated for each farm and filled completely with the prepared bedding from the respective farm. The filled chambers were saturated with reverse osmosis water and 48 µL prepared oocyst (2 x 108 mL–1) solution (purified oocysts in reverse osmosis water) was pipetted into the saturated chamber medium. The chambers, wrapped in parafilm and stored at 4°C, were then transported to the farms for installation in the calf bedding.
A control accompanied each chamber. A control consisted of a fully enclosed 1.5 mL microcentrifuge tube containing 1 mL volume of reverse osmosis water and 1 x 106 oocysts. These tubes were allowed no air, chemical, or water transfer between the environment and the tube's interior.
Installation
On each farm, chambers and controls were orientated horizontally in two protective discs, which were situated in the bedding under the calves. The discs were designed to protect the chambers from being trampled or ingested by the calves, but still allow them to be thoroughly exposed to the surrounding environment. The protective discs were composed of a ring of polyvinyl plastic (outside diameter = 25 cm, inside diameter = 20 cm, and height = 3 cm), enclosed by two circles of stainless steel expanded metal flattened mesh (2.75- x 1.0-cm openings) and attached with stainless steel machine screws (Fig. 4
). To access the chambers and controls during sampling, one circle of the expanded metal was removed using a hand drill. A data logger (HOBO TEMP; Onset Computer Corp., Pocasset, MA) recorded hourly bedding temperatures on each of the farms. Daily average temperatures for Delhi, NY (weather station with closest proximity to all four farms) were compiled from Climate Information for Management and Operational Decisions (CLIMOD) (2002) in the Northeast Regional Climate Center database.
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Fig. 4. A protective disc securely contained both controls and chambers. Samples were removed by opening one piece of expanded metal with a hand drill.
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The discs remained on each farm for the entire duration of the study, December 2001 through August 2002. They were initially placed with calves less than 30 d of age. In SCH the discs were placed under the bedding and in the CCH the discs were placed on the floor surrounded by bedding. The bedding and calf management and the disc locations on each of the farms are summarized in Table 1.
Sampling
The study period, December 2001 to August 2002, was divided into four 2-mo-long sampling seasons (winter, spring, summer, and late summer). At the beginning of each season, chambers and controls were secured in the discs and situated in the calf bedding. After 4, 6, and 8 wk, a chamber and control pair were removed from each of the discs for analysis. To investigate the effects of longer environmental exposure, sets of chamber and control pairs were installed at the beginning of the spring and summer seasons and remained in the discs until the end of the late summer season (6 and 4 mo, respectively).
Analysis
The viability of the oocysts in the chambers and the controls was routinely determined by a modified version of a dye permeability assay (Kato et al., 2001a), previously described by Jenkins et al. (1997, 1999). Oocysts were extracted from the chambers and controls by sucrose-gradient extraction and stained with propidium iodide (PI) [1 mg mL–1 in 0.1 M phosphate-buffered saline (PBS)]. The oocysts were then labeled with monoclonal antibodies specific to C. parvum oocysts and a fluorescein isothiocyanate (FITC)–conjugated antibody (Hydrofluor-Combo; Strategic Diagnostics, Newark, DE) specific to products of the reaction between monoclonal antibody and the surfaces of oocysts.
Microscopy
All samples were examined with Nikon (Tokyo, Japan) Eclipse E600 in differential interference contrast (DIC) and epifluorescence equipped with a filter block optimized for FITC counterstained with PI; excitation wavelength from 450 to 490 nm (nanometers), dichroic mirror wavelength of 500 nm, and barrier wavelength of 515 nm. Inactivation of oocysts was indicated by the migration of PI into the oocyst wall (PI+). Empty oocysts were also considered inactive. Empty oocysts result when oocysts break open and the infectious agents (sporozoites) are consequently exposed to environment. A minimum of 100 oocysts were counted and categorized as PI+ (inactive), PI– (viable), or empty (inactive) in one sample. Percent viability was determined by dividing the number of viable oocysts by the sum of oocysts counted.
Statistical Analysis
A mixed model analysis was performed in SAS (SAS Institute, 1999) to investigate the interactions of fixed variables, such as housing type, sampling interval, and treatment (control vs. chamber), and the random effect of farm type on arcsine-transformed viability data during each sampling season. Interactions resulting in probability values (P value) of less than 0.05 were considered significant. All other statistical analyses of data were performed in Minitab statistical software (Minitab, 2000).
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RESULTS
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The overall viability of the extracted oocysts from each of the chambers and the controls collected was used to calculate the percent viability. Zero percent viability indicated all oocysts in a sample were inactive, or dead. The PI– oocysts were considered viable and PI+ and empty oocysts were considered inactive. Several samples contained empty (excysted) oocysts (Table 2). Chamber samples contained a significantly (P > 0.05) greater percentage of empty oocysts than controls. For the winter and spring seasons, very few empty oocysts were observed in the controls, but there was a substantial increase of empty oocysts reported in the controls during the summer and especially during the late summer.
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Table 2. Summary of excysted (empty) oocysts in control and chamber samples throughout each sampling season. Propidium iodide (PI– and PI+) oocysts make up the remaining percentage of oocysts.
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In addition to viabilities for each farm, bedding temperatures and outdoor temperatures were compiled and compared. The winter (December to March) of 2001–2002 was mild with only 62 d in which average daily air temperatures were below 0°C. The bedding in both CCH never reached 0°C, while average daily bedding temperatures in both SCH were below freezing for 7 (Farm A103) and 16 (Farm C304) days. Although the calf housing facilities were protected from the colder outdoor temperatures, the temperature fluctuations in the bedding coincided with the fluctuations in outdoor temperature recorded at Delhi (Table 3). However, there was a significant (P > 0.05) difference between outdoor temperatures and the bedding temperatures in the calf housing facilities and no significant (P > 0.05) difference between the bedding temperatures among the facilities. Greater ranges (32.0 and 31.1°C) of bedding temperatures were observed in SCH compared to the ranges (20.1 and 24.0°C) in CCH. Furthermore, SCH maintained colder bedding temperatures during the winter and early spring seasons and warmer temperatures in the summer season relative to CCH.
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Table 3. Summary of daily average temperature data recorded in the calf bedding on each farm and at the weather station in Delhi, NY (Delhi 2 SE) from CLIMOD. Range of values is given first followed by average over the period in parentheses.
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In Fig. 5
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the mean inactivation rate of both control and chamber oocysts are presented for each of the calf housing facilities. The individual points in the graphs represent the actual percent viabilities observed for each chamber and control at each sampling. Inactivation of both chamber and control oocysts was less during the winter compared to the three sampling seasons in the warmer months (April to August 2002). During the first, or winter, season, chamber oocyst mean viability decreased to as low as 28.5, 14.5, 28.5, and 29.0% for Farms A103, C304, A105, and B205, respectively. Mean percent viability decreased to less than 20% for each of the remaining seasons on all the farms.
During the first three seasons (December 2001 to June 2002), the inactivation of control oocysts was significantly less than chamber oocysts, between 20 and 30% less. During the final, or late summer, season when bedding temperatures increased above 15°C, the percent viability of the control oocysts decreased more than the chamber oocysts, resulting in mean percent viabilities of 0 to 1% in controls and 3.0 to 23.8% in chambers. The comparison of oocyst survival in the two different types of housing facilities (SCH and CCH) is presented in Fig. 6
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The inactivation of oocysts in the chambers is represented by solid lines and by dashed lines for controls. Sample results from Farm A103 and C304 represented SCH (dark colored lines) and Farm A105 and B205 represented CCH (light colored lines). There was no significant (P > 0.05) difference in the mean percent viabilities between the SCH and CCH.
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Fig. 6. Comparison of inactivation rates for chamber (solid lines) and control (dashed lines) oocysts in solar (dark color) and main barn (light color) calf housing facilities. Mean hourly bedding temperature (dotted lines) also provided.
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DISCUSSION
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The sentinel chambers proved effective in comparing the survival of C. parvum in two types of calf housing facilities. Although reported as a conservative estimate (Kato et al., 2001a), the dye permeability assay employed to assess the viability of sample oocysts provided reliable results in this study. Three categories of oocysts, PI+, PI–, and empty, were readily identified. Empty oocysts may have been responsible for the anomalous increases in percent viability observed during the last sampling of some seasons (Fig. 5). The extraction process used to collect the oocysts, which are then dyed with PI, may explain this. Specifically, oocysts were concentrated and extracted by centrifuging and suspending oocysts in solutions of varying specific gravities. Empty and broken oocysts have specific gravities different from that of intact oocysts and, therefore, empty oocysts would not have effectively been suspended and may have been lost during this process. The remaining oocysts would not fully represent these originally present in the sample, thus skewing the percent viable counts. In Kato and Bowman (2002), a fluorescent-activated cell sorter was also unable to effectively account for excysted oocysts when reading PI-stained and FITC-labeled oocysts (dye permeability assay) because the appearance (i.e., size and detectable fluorescence) of the oocyst was different from those of intact oocysts.
Chamber samples had significantly (P > 0.05) more excysted (empty) oocysts compared to control samples (Table 2). Because controls were secured from the environment, except with respect to temperature, the controls were protected from some environmental factors that caused the oocysts to become inactive or excyst in the chambers. For example, nontemperature factors that may have influenced oocyst inactivation and excystation within calf bedding include desiccation and the presence of free ammonia. Although these factors were not investigated in this study, Robertson et al. (1992) showed experimentally that desiccation successfully inactivated 100% of the exposed oocysts within 4 h while Jenkins et al. (1998) found that ammonia exposure increased the permeability of the oocyst wall making the internal structures more susceptible to external stresses. During the dry, warm weather and with the addition of clean bedding, bedding packs may dry enough to create conditions in which desiccation can occur and affect oocyst viability. Since clean straw was added on the used bedding, calf bedding at all the farms remained under the calves for extended periods (>1 d and sometimes as many as 90 d). The increased depth of bedding and the time it remains under the calves has resulted in substantial ammonia (NH3) emission, increasing to over 900 mg m–2 h–1 during an 8-wk period in bedding of long straw (Jeppsson, 1999). One overriding factor may not be responsible for inactivation but a combination of these factors (temperature, desiccation, and ammonia) has the potential to inactivate oocysts within the bedding.
In soil, freeze–thaw and extended freezing cycles significantly decreased oocyst viability (Kato and Bowman, 2002; Jenkins et al., 1999) and caused many of the oocysts to deform and excyst (Jenkins et al., 1999). Although the same may be true for oocysts in bedding, the mild winter in 2001–2002 produced very few freezing temperatures in the calf bedding to fully substantiate the effect of freezing, and freezing temperatures were only recorded in the solar calf houses. The mild temperatures were conducive to the survival of control oocysts as indicated by the difference between chamber and control inactivation. However, in the late summer sampling season, bedding temperatures remained above 15°C, and inactivation of control oocysts was actually more substantial than the inactivation of chamber oocysts. Bedding temperature may only have been one of a combination of previously discussed factors affecting the inactivation of chamber oocysts.
Although differences in oocyst inactivation rates between SCH and CCH were anticipated, we observed no significant (P > 0.05) difference in oocyst survival between the two types of housing facilities (Fig. 6). Therefore, if there are any differences in the risk of C. parvum contamination from the two housing strategies, it will involve differences in the bedding management in each of the facilities. Consider that after 6 wk (42 d) in the bedding, the oocyst viability significantly decreased in both types of housing facilities. In the CCH, soiled bedding was removed and field applied weekly and if the bedding was contaminated with oocysts, a large proportion of them would likely be viable when spread on the fields. On the contrary, bedding remained for several weeks in SCH, then it was piled, and eventually, >6 wk later, applied to the fields. Conceivably, the SCH manure would have few viable oocysts when it was spread on the fields. Furthermore, the calf manure in the SCH can be more easily isolated from the main herd waste and, thus, facilitate additional treatment to the manure most at risk of contamination.
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CONCLUSIONS
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Although oocyst inactivation was similar in both solar and conventional calf housing facilities, the specific management practices employed in SCH may have a greater influence on oocyst control. Solar calf housing allowed calf-bedding waste to be easily retained for extended periods of time, isolated from the main herd, and stored before field application. On farms without solar calf housing, alternatives may be available for effective calf waste management and the control of C. parvum in the watershed. For example, the calf bedding waste removed from the calf-holding area of those calves under the age of 1 mo could be separated from the rest of the barn waste, stored, and composted before field spreading.
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ACKNOWLEDGMENTS
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The authors would like to express their gratitude to the Watershed Agricultural Council (WAC) and Watershed Agricultural Program (WAP) for financial support and assistance and especially to the families on each of the participating farms for their patience and cooperation. We would also like to extend special thanks to Betty Czarniecki for editing and formatting and Francoise Vermeylen for helpful statistical advice.
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ABSTRACT
INTRODUCTION
