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Giardia duodenalis cyst survival in cattle slurry
Veterinary Parasitology 184 (2012) 330–334
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Giardia duodenalis cyst survival in cattle slurry G.H. Grit a,∗ , E. Bénéré b , A. Ehsan a , N. De Wilde a , E. Claerebout a , J. Vercruysse a , L. Maes b , T. Geurden a a b
Laboratory for Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Laboratory of Microbiology, Parasitology & Hygiene (LMPH), University of Antwerp, 2020 Antwerp, Belgium
a r t i c l e
i n f o
Article history: Received 14 February 2011 Received in revised form 9 August 2011 Accepted 11 August 2011 Keywords: Giardia duodenalis Cattle Slurry Cyst survival Infectivity
a b s t r a c t Giardia duodenalis is a protozoan parasite known to infect animals and humans. Zoonotic transmission of G. duodenalis can occur by the consumption of drinking water produced from surface water that is contaminated by runoff from manure-laden fields or pastures. Although it was previously reported that storing solid cattle manure decreases G. duodenalis cyst viability, no data are available on cyst survival in slurry waste from cattle. In this study the number and the viability of G. duodenalis cysts was determined in cattle slurry for up to 90 days. G. duodenalis cysts were counted in 30 slurry samples with a quantitative direct immunofluorescence assay. The geometric mean number of cysts was reduced by 77% after 90 days (P < 0.0014), although there was substantial variability between samples. A fluorogenic dye staining using 4 ,6 -di-amino-2-phenylindole and propidium iodide showed a decreased viability from 45 days onwards, and after 90 days incubation, only 3% of the cysts were viable. Gerbils and lambs were artificially infected with 50 day-old and 90 dayold cysts and faecal excretion of G. duodenalis was monitored between 5 and 7 days after infection. Seven days after infection the gerbils were euthanized for Giardia trophozoite counts. Although one cyst was found in the faeces of one of the gerbils after infection with 50 day-old cysts, no trophozoites were recovered from the intestines of any gerbil (n = 8). Experimental infection of lambs with 105 50 day-old and 90 day-old slurry cysts caused low cyst excretion in one out of two and one out of three lambs, respectively. Together, these data show that storage of cattle slurry for 90 days greatly reduces the number and viability of G. duodenalis cysts. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Giardia duodenalis is frequently seen in cattle and causes intermittent diarrhoea and weight loss in young animals (Geurden et al., 2009, 2010). In humans, G. duodenalis is the most frequently occurring parasitological cause of diarrhoea, with an estimated 200 million cases each year worldwide (Savioli et al., 2006). G. duodenalis is a species complex comprising 8 different assemblages, which are either zoonotic or host-specific. In cattle both the zoonotic assemblage A and the livestock-specific assemblage E have
∗ Corresponding author. Tel.: +32 0 9 2647516; fax: +32 0 9 2647496. E-mail address: [email protected] (G.H. Grit). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.08.021
been identified (Geurden et al., 2008). G. duodenalis has a direct lifecycle, and large numbers of cysts are excreted into the environment via the faeces. Zoonotic transmission can occur through direct contact or indirectly via contaminated food or water (Castro-Hermida et al., 2009). Especially water is an important source, and giardiasis is one of the most commonly reported waterborne infections worldwide (Thompson, 2004). Surface water contamination can occur through direct contamination by cattle, manurecontaminated run-off water from farms or the application of animal waste to agricultural land (Schijven et al., 2004). The potential for environmental contamination is largely defined by the release efficiency of cysts from the faeces (Schijven et al., 2004) and the survival time of the cysts, which depends on different factors, such as moisture,
G.H. Grit et al. / Veterinary Parasitology 184 (2012) 330–334
exposure to sunlight, ambient temperature, pH and salinity (Olson et al., 1999). Furthermore, the cyst survival depends to a large extent on the matrix in which the cysts remain: in experimental assays Giardia cysts survive up to 12 weeks in water (Brookes et al., 2004; deRegnier et al., 1989; Olson et al., 1999, 2004; Robertson and Gjerde, 2006) and up to 7 weeks in soil (Olson et al., 1999). In faecal material the cyst survival time is limited to 18 days in human faeces (Deng and Cliver, 1992; Thiriat et al., 1998) and only 7 days in solid cattle faeces (Olson et al., 1999). To date, stables with solid floors covered with bedding material such as straw and sawdust are often replaced by housing with slatted floors. As a result the physical nature of the manure changes from solid manure to more liquid slurry. Cysts present in liquid slurry waste may have a higher chance of survival because they are not exposed to diurnal temperature fluctuations, UV radiation and the desiccating effects of air flows (Hutchison et al., 2005). In the present study, the survival and viability of G. duodenalis cysts in calf slurry was examined. The reduction in G. duodenalis cyst numbers over time in cattle slurry was determined using an immunofluorescence assay (IFA). The viability was monitored by 4 ,6 -di-amino-2-phenylindol (DAPI) and propidium iodide (PI) staining. To confirm these in vitro results, gerbils and sheep were infected with 50 and 90 days old cysts and cyst excretion was monitored until 7 days after infection using the IFA. 2. Materials and methods 2.1. Experimental slurry samples Thirty fresh faecal samples from 1 to 3 months old calves with a natural G. duodenalis infection were diluted 1:1 (v/v) with distilled water and thoroughly mixed. These experimental slurry samples were kept in darkness, under micro-aerobic conditions at a temperature range of 14–18 ◦ C in order to mimic natural slurry storage conditions. The number of cysts in the initial faecal sample was not a criterion for selection, and samples with a broad range of cyst concentrations (cysts per gram faeces) were included. 2.2. Quantification of Giardia cysts in slurry samples The number of G. duodenalis cysts was determined in 30 slurry samples every fortnight during 90 days. A 1 ml aliquot was taken at every sampling time after thoroughly mixing the slurry sample. The number of cysts per gram faeces (CPG) was determined using a quantitative direct immunofluorescence assay (IFA) based on the commercial MERIFLUOR Cryptosporidium/Giardia kit (Meridian Diagnostics Inc., Cincinnati, Ohio), as described by Geurden et al. (2004). Cyst counts on each sampling day were compared with D0 cyst counts using a one-tailed Mann–Whitney test, and P values <0.05 were considered to indicate significant differences. 2.3. Fluorogenic dye staining In a second experiment, the cyst viability was determined in six slurry samples on day 45, 75 and 90, by staining
331
with propidium iodide in combination with indirect fluorescent antibody detection, based on previous protocols (Dowd and Pillai, 1997). After fluorescein isothiocyanate (FITC) staining, 50 l of 4 ,6 -di-amino-2-phenylindole (DAPI di-lactate, Invitrogen (Molecular Probes) cat. nr. D3571, 0.004 mg/ml methanol) and 5 l propidium iodide Hoechst solution (PI, Invitrogen (Molecular Probes), cat. nr. P1304MP, 100 mg) was added and incubated in the dark. After the staining, the microscope slide was washed with PBS to remove the DAPI and PI and prepared with mounting medium containing glycerine-DABCO*2 (Acros Organics: cat. nr. 112471000). The slide was covered and read using a fluorescent microscope with a FITC/TRITC filter (450–590 nm) and a Texas Red filter (530–585 nm). Counts of viable cysts on the consecutive sampling days were compared with counts on day 0 using a one-tailed Mann–Whitney test, and P values <0.05 were considered to indicate significant differences. 2.4. Gerbil infection experiment Two groups of four gerbils (Meriones unguiculatus) were infected with G. duodenalis cysts, maintained under the same conditions as described above for 50 (D50) and 90 (D90) days, respectively. Eight 6 week-old male gerbils purchased from Janvier (Le Genest St. Isle, France) were housed individually and fed commercial pellets and water ad libitum for an acclimatisation period of 1 week. Before experimental infection, previous contact with Giardia was excluded by microscopic examination of the faeces collected for 3 consecutive days using IFA. The animals were fasted overnight before oral inoculation with 1 × 106 Giardia cysts (Bénéré et al., 2010) originating from a naturally infected calf. In order to prepare the infective dosages, faeces from a naturally infected calf with Giardia, were kept under the same conditions as described above for 50 (D50) and 90 days (D90). Five days after infection, faeces were collected from the infected gerbils and Giardia cysts were counted as described above. Seven days after infection the gerbils were fasted overnight, euthanized and the entire small intestine was aseptically removed, opened longitudinally and suspended in phosphate buffered saline (PBS). Trophozoites were isolated from the small intestine using a modified method (Bénéré et al., 2010), in order to determine the presence of duodenal trophozoites. 2.5. Lamb infection experiment Two groups of 2 and 3 lambs aged 6 months were infected with 50 days old and 90 days old Giardia cysts, respectively. The lambs were housed on straw bedding in two separate pens that were thoroughly cleaned and disinfected with ammonia 10% (Geurden et al., 2006). Previous contact with Giardia was excluded by microscopic examination of the faeces collected every three days, during a period of 9 days, using IFA. As for the gerbil experiment, infection doses were prepared with Giardia cysts from slurry that had been stored for 50 and 90 days. The total infection dose for each lamb was 1 × 105 cysts (Geurden et al., 2011). The cyst excretion was monitored on days 5 and 7 after infection, using IFA.
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4. Discussion
cysts per gram
1000 800 600 400 200 0 d0
d15
d30
d45
d60
d75
d90
time in days Fig. 1. The geometric mean number of Giardia duodenalis cysts per gram of faeces for the 30 slurry samples, on each sampling day (d).
3. Results 3.1. Quantification and viability of Giardia cysts The cyst counts showed large fluctuations between the different samples and within samples over time (from 0 to 3.7 × 104 cysts per gram, Appendix A). Despite this large variability, the geometric mean cyst counts on D90 were 77% lower compared to D0 (P < 0.0014) (Fig. 1 and Appendix A). Fig. 2 shows the decrease of the percentage viable cysts from 99.8% on day 0–3.1% on day 90. The viability of the cysts was significantly reduced (P < 0.037) from D45. 3.2. Gerbil infection experiment Except for one gerbil, excreting 50 cysts per gram faeces on day 5, none of the gerbils with 50 day-old cysts, excreted Giardia cysts five days after infection. No Giardia cysts were detected in faeces from the gerbils that were given 90 dayold cysts. No trophozoites were found in the small intestine in any of the gerbils after euthanasia. 3.3. Lamb infection experiment
Percentage of viabel cysts
No cysts were detected in the faeces collected 5 days after infection of 5 lambs with either 50 day-old or 90 dayold cysts. On day 7 after infection, one lamb infected with 50 day-old cysts and one lamb infected with 90 day-old cysts became positive, with counts of 100 CPG and 2200 CPG, respectively.
120 100 80 60 40 20 0 D0
D45
D75
D90
Time in days Fig. 2. The percentage of viable Giardia duodenalis cysts (±stdev) in function of time.
The present study aimed to investigate the survival of G. duodenalis cysts in cattle slurry. In a previous study, Olson et al. (1999) reported a survival time of G. duodenalis cysts of only one week in solid cattle faeces. However, an increasing number of cattle farms invest in slatted flooring without or with a limited use of solid bedding material. As a result, the consistency of the cattle manure changes from a solid to a slurry type of manure. This change in matrix and consequently the waste management at farm level might influence the survival of G. duodenalis cysts, and hence the potential for environmental contamination. In another study, the Giardia cyst survival in human septic tank effluent, mixed or not with swine slurry (Deng and Cliver, 1992), was longer compared to cyst survival time in solid cattle manure (Olson et al., 1999). Although other parameters might have contributed to the differences between both studies, these results seem to suggest that G. duodenalis cyst survival is potentially longer in slurry than in solid manure, due to the more suitable conditions for cyst survival. In the present study there was a substantial variability in cyst counts between samples and between different time points within samples. In some samples the number of cysts even increased over time. This variability could be due to clustering of the cysts in the faeces, the small volume of the aliquots that were investigated with the IFA and/or changes in the faecal consistency over the experimental period, which may facilitate the release of cysts from the faecal matrix (Schijven et al., 2004; Hutchison et al., 2005). Despite the high variability of the cyst counts, the number of cysts decreased significantly by D90. Moreover DAPI/PI staining demonstrated that the vast majority of the cysts that were present after 90 days incubation were no longer viable. Some authors stated that the viability rate found by PI staining is even an overestimation of the true cyst viability (Smith and Smith, 1989). These results suggest that G. duodenalis cysts do not immediately disintegrate after inactivation and that detection of cysts without assessment of their viability is not a reliable parameter to monitor infection pressure and risk of transmission. Therefore, two experiments were conducted to test the cyst infectivity in vivo. The gerbil was previously shown to be a good infection model for bovine G. duodenalis isolates (Bénéré et al., 2010). Infection with cysts that were incubated for 50 and 90 days in cattle slurry failed to induce a patent infection in all but one of the gerbils. These results indicate a low viability and infectivity of the slurry cysts since similar infections with fresh cysts from cattle using a similar infection route and dosage yielded a high infection rate in previous experiments (Bénéré et al., 2010). An experimental infection in lambs was used to confirm the results obtained in the gerbils. Although one of the two animals infected with 50 day-old cysts and one out of three lambs infected with 90 day-old cysts were excreting cysts after infection, the results suggest that the infectivity of the incubated cysts was low, as not all animals excreted cysts and as the cyst excretion was quite low after infection. In contrast, high infectivity rates and cyst excretions were observed after experimental infection with 105 fresh
G.H. Grit et al. / Veterinary Parasitology 184 (2012) 330–334
cysts in calves (Geurden et al., 2010) and lambs (Geurden et al., 2011). Previous studies indicated that Giardia cysts survive for several weeks to months in water (deRegnier et al., 1989; Brookes et al., 2004; Olson et al., 2004; Robertson and Gjerde, 2006), and these observations alarmed researchers and public health authorities on the potential threat of infection. Work by Olson et al. (1999) indicated that keeping the manure for a couple of weeks might be a good measure to decrease cyst infectivity in cattle faeces, but manure and slurry spreading were still considered as potential infection sources for the environment. Although slurry can be considered to be a more suitable matrix for cyst survival compared to solid manure, the results of the present study indicate that storing slurry for 50–90 days significantly reduces the number of cysts and the cyst viability, and that the infectivity of the cysts is strongly reduced. Furthermore, in natural conditions, the presence of ammonia (urine) in slurry may further decrease the cyst survival and infectivity. Further studies are needed to investigate how long slurry should be stored under natural conditions to avoid transmission of viable Giardia cysts into the environment.
Acknowledgements We thank Sisca Schets and colleagues of the RIVM (Bilthoven, The Netherlands) for sharing their expertise on viability staining. We are grateful to Stijn Casaert for technical assistance. This research was funded by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). E. Bénéré has a PhD grant from the FWO Vlaanderen.
Appendix A. The number of G. duodenalis cysts per gram in each experimental slurry sample, along with the reduction over the 90 days experimental period (red D0–D90), as calculated for each sample. No. D0 R1 450 R2 475 R3 525 R4 19,625 R5 24,400 R6 500 R7 1250 R8 6800 R9 13,300 R10 8600 R10 650 R12 250 R13 650 R14 515 R15 500 R17 625 R18 200 R19 6300
D15 200 450 500 6450 8750 4250 2100 9100 18,450 20,050 100 150 1000 350 450 700 350 2100
D30 150 750 500 17,400 4200 3900 1350 2750 15,400 13,550 200 200 850 250 150 700 150 2300
D45
D60
350 300 1050 2900 300 400 16,800 17,800 2150 8750 1750 650 700 0 5250 1500 8400 450 9750 1200 0 200 100 50 650 200 100 50 200 250 600 350 0 450 2500 1500
D75 100 1500 450 14,700 15,900 4300 1850 7450 29,600 31,350 750 150 550 150 200 150 350 1250
D90
Red D0–D90
350 22% 1700 0% 400 24% 36,850 0% 14,020 43% 1200 0% 1050 16% 5000 26% 13,250 0% 14,500 0% 100 85% 0 100% 100 85% 100 81% 0 100% 250 60% 0 100% 50 99%
No. D0 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30
D15 250 625 450 350 350 200 250 500 450 250 500
500 450 350 150 250 200 250 150 100 100 200
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D30 100 400 100 0 50 0 50 550 100 250 200
D45 250 350 250 250 300 100 100 550 50 100 250
D60 50 350 50 100 100 150 0 300 50 200 50
D75 200 450 0 300 250 150 200 150 50 200 350
D90 0 250 0 0 0 100 50 50 0 50 0
Red D0–D90 100% 60% 100% 100% 100% 50% 80% 90% 100% 80% 100%
References Bénéré, E., Geurden, T., Robertson, L., Van Assche, T., Cos, P., Maes, L., 2010. Infectivity of Giardiaduodenalis assemblage A and E for the gerbil and axenisation of duodenal trophozoites. Parasitol. Int. 59, 634–637. Brookes, J.D., Antenucci, J., Hipsey, M., Burch, M.D., Ashbolt, N.J., Ferguson, C., 2004. Fate and transport of pathogens in lakes and reservoirs. Environ. Int. 30, 741–759. Castro-Hermida, J.A., García-Presedo, I., Almeida, A., González-Warleta, M., Da Costa, J.M.C., Mezo, M., 2009. Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a health risk for humans and animals. Water Res. 43, 4133–4142. Deng, M.Y., Cliver, D.O., 1992. Degradation of Giardia lamblia cysts in mixed human and swine wastes. Appl. Environ. Microbiol., 368–2374. deRegnier, D.P., Cole, L., Schupp, D.G., Erlandsen, S.L., 1989. Viability of Giardia cysts suspended in lake, river, and tab water. Appl. Environ. Microbiol. 55 (5), 1223–1229. Dowd, S.E., Pillai, S.D., 1997. Survival and transport of selected bacterial pathogens and indicator viruses under sandy aquifer conditions. J. Environ. Sci. Health 32, 2245–2258. Geurden, T., Claerebout, E., Vercruysse, J., Berkvens, D., 2004. Estimation of diagnostic test characteristics and prevalence of Giardia duodenalis in dairy calves in Belgium using a Bayesian approach. Int. J. Parasitol. 34, 1121–1127. Geurden, T., Vercruysse, J., Claerebout, E., 2006. Field testing of a fenbendazole treatment combined with hygienic and management measures against a natural Giardia infection in calves. Vet. Parasitol. 142, 367–371. Geurden, T., Geldhof, P., Levecke, B., Martens, C., Berkvens, D., Casaert, S., Vercruysse, J., Claerebout, E., 2008. Mixed Giardia duodenalis assemblage A and E infections in calves. Int. J. Parasitol. 38, 259–264. Geurden, T., Vercruysse, J., Claerebout, E., 2009. Is Giardia a significant pathogen in production animals? Exp. Parasitol. 124, 98–106. Geurden, T., Vandenhoute, E., Pohle, H., Casaert, S., De Wilde, N., Vercruysse, J., Claerebout, E., 2010. The effect of a fenbendazole treatment on cyst excretion and weight gain in calves experimentally infected with Giardia duodenalis. Vet. Parasitol. 169, 18–23. Geurden, T., Pohle, H., Sarre, C., Dreesen, L., Vercruysse, J., Claerebout, E., 2011. The efficacy of a treatment with fenbendazole against an experimental Giardia duodenalis infection in lambs. Small Ruminant Res. 96, 211–215. Hutchison, M.L., Walters, L.D., Moore, T., Thomas, D.J.I., Avery, S.M., 2005. Fate of pathogens present in livestock wastes spread onto fescue plots. Appl. Environ. Microbiol. 71, 691–696. Olson, M.E., O’Handley, R.M., Ralston, B.J., McAllister, T.A., Thompson, R.C.A., 2004. Update on Cryptosporidium and Giardia infections in cattle. Trends Parasitol. 20, 185–191. Olson, M.E., Goh, J., Philips, M., Gunselle, N., McAllister, T.A., 1999. Giardia cyst and Cryptosporidium oocyst survival in water, soil and cattle feces. J. Environ. Qual. 28, 1991–1996. Robertson, L.J., Gjerde, B.K., 2006. Fate of Cryptosporidium oocysts and Giardia cysts in the Norwegian aquatic environment over winter. Microb. Ecol. 52, 597–602. Savioli, L., Smith, H., Thompson, A., 2006. Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol. 22, 203–208. Schijven, J.F., Bradford, S.A., Yang, S., 2004. Release of Cryptosporidium and Giardia from dairy cattle manure: physical factors. J. Environ. Qual. 33, 1499–1508. Smith, A.L., Smith, H.V., 1989. A comparison of fluorescein diacetate and propidium iodide staining and in vitro excystation for determining Giardia intestinalis cyst viability. Parasitology 99, 329–331.
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Thiriat, L., Sidaner, F., Schwarzbrod, J., 1998. Determination of Giardia cyst viability in environmental and faecal samples by immunofluorescence, fluorogenic dye staining and differential interference contrast microscopy. Lett. Appl. Microbiol. 26, 237–242.
Thompson, R.C.A., 2004. The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Vet. Parasitol. 126, 15–35.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Giardia duodenalis cyst survival in cattle slurry G.H. Grit a,∗ , E. Bénéré b , A. Ehsan a , N. De Wilde a , E. Claerebout a , J. Vercruysse a , L. Maes b , T. Geurden a a b
Laboratory for Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Laboratory of Microbiology, Parasitology & Hygiene (LMPH), University of Antwerp, 2020 Antwerp, Belgium
a r t i c l e
i n f o
Article history: Received 14 February 2011 Received in revised form 9 August 2011 Accepted 11 August 2011 Keywords: Giardia duodenalis Cattle Slurry Cyst survival Infectivity
a b s t r a c t Giardia duodenalis is a protozoan parasite known to infect animals and humans. Zoonotic transmission of G. duodenalis can occur by the consumption of drinking water produced from surface water that is contaminated by runoff from manure-laden fields or pastures. Although it was previously reported that storing solid cattle manure decreases G. duodenalis cyst viability, no data are available on cyst survival in slurry waste from cattle. In this study the number and the viability of G. duodenalis cysts was determined in cattle slurry for up to 90 days. G. duodenalis cysts were counted in 30 slurry samples with a quantitative direct immunofluorescence assay. The geometric mean number of cysts was reduced by 77% after 90 days (P < 0.0014), although there was substantial variability between samples. A fluorogenic dye staining using 4 ,6 -di-amino-2-phenylindole and propidium iodide showed a decreased viability from 45 days onwards, and after 90 days incubation, only 3% of the cysts were viable. Gerbils and lambs were artificially infected with 50 day-old and 90 dayold cysts and faecal excretion of G. duodenalis was monitored between 5 and 7 days after infection. Seven days after infection the gerbils were euthanized for Giardia trophozoite counts. Although one cyst was found in the faeces of one of the gerbils after infection with 50 day-old cysts, no trophozoites were recovered from the intestines of any gerbil (n = 8). Experimental infection of lambs with 105 50 day-old and 90 day-old slurry cysts caused low cyst excretion in one out of two and one out of three lambs, respectively. Together, these data show that storage of cattle slurry for 90 days greatly reduces the number and viability of G. duodenalis cysts. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Giardia duodenalis is frequently seen in cattle and causes intermittent diarrhoea and weight loss in young animals (Geurden et al., 2009, 2010). In humans, G. duodenalis is the most frequently occurring parasitological cause of diarrhoea, with an estimated 200 million cases each year worldwide (Savioli et al., 2006). G. duodenalis is a species complex comprising 8 different assemblages, which are either zoonotic or host-specific. In cattle both the zoonotic assemblage A and the livestock-specific assemblage E have
∗ Corresponding author. Tel.: +32 0 9 2647516; fax: +32 0 9 2647496. E-mail address: [email protected] (G.H. Grit). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.08.021
been identified (Geurden et al., 2008). G. duodenalis has a direct lifecycle, and large numbers of cysts are excreted into the environment via the faeces. Zoonotic transmission can occur through direct contact or indirectly via contaminated food or water (Castro-Hermida et al., 2009). Especially water is an important source, and giardiasis is one of the most commonly reported waterborne infections worldwide (Thompson, 2004). Surface water contamination can occur through direct contamination by cattle, manurecontaminated run-off water from farms or the application of animal waste to agricultural land (Schijven et al., 2004). The potential for environmental contamination is largely defined by the release efficiency of cysts from the faeces (Schijven et al., 2004) and the survival time of the cysts, which depends on different factors, such as moisture,
G.H. Grit et al. / Veterinary Parasitology 184 (2012) 330–334
exposure to sunlight, ambient temperature, pH and salinity (Olson et al., 1999). Furthermore, the cyst survival depends to a large extent on the matrix in which the cysts remain: in experimental assays Giardia cysts survive up to 12 weeks in water (Brookes et al., 2004; deRegnier et al., 1989; Olson et al., 1999, 2004; Robertson and Gjerde, 2006) and up to 7 weeks in soil (Olson et al., 1999). In faecal material the cyst survival time is limited to 18 days in human faeces (Deng and Cliver, 1992; Thiriat et al., 1998) and only 7 days in solid cattle faeces (Olson et al., 1999). To date, stables with solid floors covered with bedding material such as straw and sawdust are often replaced by housing with slatted floors. As a result the physical nature of the manure changes from solid manure to more liquid slurry. Cysts present in liquid slurry waste may have a higher chance of survival because they are not exposed to diurnal temperature fluctuations, UV radiation and the desiccating effects of air flows (Hutchison et al., 2005). In the present study, the survival and viability of G. duodenalis cysts in calf slurry was examined. The reduction in G. duodenalis cyst numbers over time in cattle slurry was determined using an immunofluorescence assay (IFA). The viability was monitored by 4 ,6 -di-amino-2-phenylindol (DAPI) and propidium iodide (PI) staining. To confirm these in vitro results, gerbils and sheep were infected with 50 and 90 days old cysts and cyst excretion was monitored until 7 days after infection using the IFA. 2. Materials and methods 2.1. Experimental slurry samples Thirty fresh faecal samples from 1 to 3 months old calves with a natural G. duodenalis infection were diluted 1:1 (v/v) with distilled water and thoroughly mixed. These experimental slurry samples were kept in darkness, under micro-aerobic conditions at a temperature range of 14–18 ◦ C in order to mimic natural slurry storage conditions. The number of cysts in the initial faecal sample was not a criterion for selection, and samples with a broad range of cyst concentrations (cysts per gram faeces) were included. 2.2. Quantification of Giardia cysts in slurry samples The number of G. duodenalis cysts was determined in 30 slurry samples every fortnight during 90 days. A 1 ml aliquot was taken at every sampling time after thoroughly mixing the slurry sample. The number of cysts per gram faeces (CPG) was determined using a quantitative direct immunofluorescence assay (IFA) based on the commercial MERIFLUOR Cryptosporidium/Giardia kit (Meridian Diagnostics Inc., Cincinnati, Ohio), as described by Geurden et al. (2004). Cyst counts on each sampling day were compared with D0 cyst counts using a one-tailed Mann–Whitney test, and P values <0.05 were considered to indicate significant differences. 2.3. Fluorogenic dye staining In a second experiment, the cyst viability was determined in six slurry samples on day 45, 75 and 90, by staining
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with propidium iodide in combination with indirect fluorescent antibody detection, based on previous protocols (Dowd and Pillai, 1997). After fluorescein isothiocyanate (FITC) staining, 50 l of 4 ,6 -di-amino-2-phenylindole (DAPI di-lactate, Invitrogen (Molecular Probes) cat. nr. D3571, 0.004 mg/ml methanol) and 5 l propidium iodide Hoechst solution (PI, Invitrogen (Molecular Probes), cat. nr. P1304MP, 100 mg) was added and incubated in the dark. After the staining, the microscope slide was washed with PBS to remove the DAPI and PI and prepared with mounting medium containing glycerine-DABCO*2 (Acros Organics: cat. nr. 112471000). The slide was covered and read using a fluorescent microscope with a FITC/TRITC filter (450–590 nm) and a Texas Red filter (530–585 nm). Counts of viable cysts on the consecutive sampling days were compared with counts on day 0 using a one-tailed Mann–Whitney test, and P values <0.05 were considered to indicate significant differences. 2.4. Gerbil infection experiment Two groups of four gerbils (Meriones unguiculatus) were infected with G. duodenalis cysts, maintained under the same conditions as described above for 50 (D50) and 90 (D90) days, respectively. Eight 6 week-old male gerbils purchased from Janvier (Le Genest St. Isle, France) were housed individually and fed commercial pellets and water ad libitum for an acclimatisation period of 1 week. Before experimental infection, previous contact with Giardia was excluded by microscopic examination of the faeces collected for 3 consecutive days using IFA. The animals were fasted overnight before oral inoculation with 1 × 106 Giardia cysts (Bénéré et al., 2010) originating from a naturally infected calf. In order to prepare the infective dosages, faeces from a naturally infected calf with Giardia, were kept under the same conditions as described above for 50 (D50) and 90 days (D90). Five days after infection, faeces were collected from the infected gerbils and Giardia cysts were counted as described above. Seven days after infection the gerbils were fasted overnight, euthanized and the entire small intestine was aseptically removed, opened longitudinally and suspended in phosphate buffered saline (PBS). Trophozoites were isolated from the small intestine using a modified method (Bénéré et al., 2010), in order to determine the presence of duodenal trophozoites. 2.5. Lamb infection experiment Two groups of 2 and 3 lambs aged 6 months were infected with 50 days old and 90 days old Giardia cysts, respectively. The lambs were housed on straw bedding in two separate pens that were thoroughly cleaned and disinfected with ammonia 10% (Geurden et al., 2006). Previous contact with Giardia was excluded by microscopic examination of the faeces collected every three days, during a period of 9 days, using IFA. As for the gerbil experiment, infection doses were prepared with Giardia cysts from slurry that had been stored for 50 and 90 days. The total infection dose for each lamb was 1 × 105 cysts (Geurden et al., 2011). The cyst excretion was monitored on days 5 and 7 after infection, using IFA.
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4. Discussion
cysts per gram
1000 800 600 400 200 0 d0
d15
d30
d45
d60
d75
d90
time in days Fig. 1. The geometric mean number of Giardia duodenalis cysts per gram of faeces for the 30 slurry samples, on each sampling day (d).
3. Results 3.1. Quantification and viability of Giardia cysts The cyst counts showed large fluctuations between the different samples and within samples over time (from 0 to 3.7 × 104 cysts per gram, Appendix A). Despite this large variability, the geometric mean cyst counts on D90 were 77% lower compared to D0 (P < 0.0014) (Fig. 1 and Appendix A). Fig. 2 shows the decrease of the percentage viable cysts from 99.8% on day 0–3.1% on day 90. The viability of the cysts was significantly reduced (P < 0.037) from D45. 3.2. Gerbil infection experiment Except for one gerbil, excreting 50 cysts per gram faeces on day 5, none of the gerbils with 50 day-old cysts, excreted Giardia cysts five days after infection. No Giardia cysts were detected in faeces from the gerbils that were given 90 dayold cysts. No trophozoites were found in the small intestine in any of the gerbils after euthanasia. 3.3. Lamb infection experiment
Percentage of viabel cysts
No cysts were detected in the faeces collected 5 days after infection of 5 lambs with either 50 day-old or 90 dayold cysts. On day 7 after infection, one lamb infected with 50 day-old cysts and one lamb infected with 90 day-old cysts became positive, with counts of 100 CPG and 2200 CPG, respectively.
120 100 80 60 40 20 0 D0
D45
D75
D90
Time in days Fig. 2. The percentage of viable Giardia duodenalis cysts (±stdev) in function of time.
The present study aimed to investigate the survival of G. duodenalis cysts in cattle slurry. In a previous study, Olson et al. (1999) reported a survival time of G. duodenalis cysts of only one week in solid cattle faeces. However, an increasing number of cattle farms invest in slatted flooring without or with a limited use of solid bedding material. As a result, the consistency of the cattle manure changes from a solid to a slurry type of manure. This change in matrix and consequently the waste management at farm level might influence the survival of G. duodenalis cysts, and hence the potential for environmental contamination. In another study, the Giardia cyst survival in human septic tank effluent, mixed or not with swine slurry (Deng and Cliver, 1992), was longer compared to cyst survival time in solid cattle manure (Olson et al., 1999). Although other parameters might have contributed to the differences between both studies, these results seem to suggest that G. duodenalis cyst survival is potentially longer in slurry than in solid manure, due to the more suitable conditions for cyst survival. In the present study there was a substantial variability in cyst counts between samples and between different time points within samples. In some samples the number of cysts even increased over time. This variability could be due to clustering of the cysts in the faeces, the small volume of the aliquots that were investigated with the IFA and/or changes in the faecal consistency over the experimental period, which may facilitate the release of cysts from the faecal matrix (Schijven et al., 2004; Hutchison et al., 2005). Despite the high variability of the cyst counts, the number of cysts decreased significantly by D90. Moreover DAPI/PI staining demonstrated that the vast majority of the cysts that were present after 90 days incubation were no longer viable. Some authors stated that the viability rate found by PI staining is even an overestimation of the true cyst viability (Smith and Smith, 1989). These results suggest that G. duodenalis cysts do not immediately disintegrate after inactivation and that detection of cysts without assessment of their viability is not a reliable parameter to monitor infection pressure and risk of transmission. Therefore, two experiments were conducted to test the cyst infectivity in vivo. The gerbil was previously shown to be a good infection model for bovine G. duodenalis isolates (Bénéré et al., 2010). Infection with cysts that were incubated for 50 and 90 days in cattle slurry failed to induce a patent infection in all but one of the gerbils. These results indicate a low viability and infectivity of the slurry cysts since similar infections with fresh cysts from cattle using a similar infection route and dosage yielded a high infection rate in previous experiments (Bénéré et al., 2010). An experimental infection in lambs was used to confirm the results obtained in the gerbils. Although one of the two animals infected with 50 day-old cysts and one out of three lambs infected with 90 day-old cysts were excreting cysts after infection, the results suggest that the infectivity of the incubated cysts was low, as not all animals excreted cysts and as the cyst excretion was quite low after infection. In contrast, high infectivity rates and cyst excretions were observed after experimental infection with 105 fresh
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cysts in calves (Geurden et al., 2010) and lambs (Geurden et al., 2011). Previous studies indicated that Giardia cysts survive for several weeks to months in water (deRegnier et al., 1989; Brookes et al., 2004; Olson et al., 2004; Robertson and Gjerde, 2006), and these observations alarmed researchers and public health authorities on the potential threat of infection. Work by Olson et al. (1999) indicated that keeping the manure for a couple of weeks might be a good measure to decrease cyst infectivity in cattle faeces, but manure and slurry spreading were still considered as potential infection sources for the environment. Although slurry can be considered to be a more suitable matrix for cyst survival compared to solid manure, the results of the present study indicate that storing slurry for 50–90 days significantly reduces the number of cysts and the cyst viability, and that the infectivity of the cysts is strongly reduced. Furthermore, in natural conditions, the presence of ammonia (urine) in slurry may further decrease the cyst survival and infectivity. Further studies are needed to investigate how long slurry should be stored under natural conditions to avoid transmission of viable Giardia cysts into the environment.
Acknowledgements We thank Sisca Schets and colleagues of the RIVM (Bilthoven, The Netherlands) for sharing their expertise on viability staining. We are grateful to Stijn Casaert for technical assistance. This research was funded by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). E. Bénéré has a PhD grant from the FWO Vlaanderen.
Appendix A. The number of G. duodenalis cysts per gram in each experimental slurry sample, along with the reduction over the 90 days experimental period (red D0–D90), as calculated for each sample. No. D0 R1 450 R2 475 R3 525 R4 19,625 R5 24,400 R6 500 R7 1250 R8 6800 R9 13,300 R10 8600 R10 650 R12 250 R13 650 R14 515 R15 500 R17 625 R18 200 R19 6300
D15 200 450 500 6450 8750 4250 2100 9100 18,450 20,050 100 150 1000 350 450 700 350 2100
D30 150 750 500 17,400 4200 3900 1350 2750 15,400 13,550 200 200 850 250 150 700 150 2300
D45
D60
350 300 1050 2900 300 400 16,800 17,800 2150 8750 1750 650 700 0 5250 1500 8400 450 9750 1200 0 200 100 50 650 200 100 50 200 250 600 350 0 450 2500 1500
D75 100 1500 450 14,700 15,900 4300 1850 7450 29,600 31,350 750 150 550 150 200 150 350 1250
D90
Red D0–D90
350 22% 1700 0% 400 24% 36,850 0% 14,020 43% 1200 0% 1050 16% 5000 26% 13,250 0% 14,500 0% 100 85% 0 100% 100 85% 100 81% 0 100% 250 60% 0 100% 50 99%
No. D0 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30
D15 250 625 450 350 350 200 250 500 450 250 500
500 450 350 150 250 200 250 150 100 100 200
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D30 100 400 100 0 50 0 50 550 100 250 200
D45 250 350 250 250 300 100 100 550 50 100 250
D60 50 350 50 100 100 150 0 300 50 200 50
D75 200 450 0 300 250 150 200 150 50 200 350
D90 0 250 0 0 0 100 50 50 0 50 0
Red D0–D90 100% 60% 100% 100% 100% 50% 80% 90% 100% 80% 100%
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