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Asymptomatic shedding of Cryptosporidium oocysts by red deer hinds and calves
Veterinary Parasitology 94 (2001) 239–246
Asymptomatic shedding of Cryptosporidium oocysts by red deer hinds and calves H.E. Skerrett∗ , C.V. Holland Department of Zoology, Trinity College, Dublin 2, Ireland Received 30 May 2000; received in revised form 25 August 2000; accepted 6 September 2000
Abstract Levels of Cryptosporidium infection in a group of red deer were monitored over a period of 1 year. Faecal samples were examined on an approximate monthly basis from adult hinds and calves for the presence of Cryptosporidium oocysts. The water–ether sedimentation method followed by sucrose flotation and a monoclonal antibody identification procedure were used. It was found that apparently healthy adult deer were shedding low numbers of oocysts in their faeces throughout the year and that there appeared to be a periparturient increase in the numbers of oocysts shed. Samples taken from 6-month-old deer calves, both in-house and on pasture, had low numbers of Cryptosporidium oocysts, indicating that the calves were also asymptomatically shedding oocysts. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium spp.; Red deer; Asymptomatic shedding; Faecal shedding
1. Introduction Cryptosporidium parvum is a coccidian, protozoan parasite that has a life cycle similar to that seen in other coccidian parasites. Excystation and an endogenous asexual stage are followed by a sexual stage resulting in the production and discharge of oocysts in the faeces (Current and Garcia, 1991). Cryptosporidium can cause a self-limiting diarrhoeal disease in immunocompetent humans and an extremely severe, life-threatening illness in the immunocompromised (Fayer and Ungar, 1986). Cryptosporidium is also one of the chief causes of diarrhoeal disease among neonatal ruminants (Angus, 1988). ∗ Corresponding author. Present address: Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. Tel.: +353-1-6687988/ext. 2606; fax: +353-1-6608656. E-mail address: [email protected] (H.E. Skerrett).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 4 0 5 - 2
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H.E. Skerrett, C.V. Holland / Veterinary Parasitology 94 (2001) 239–246
Much work has been carried out on the veterinary disease caused by Cryptosporidium, and patterns of cryptosporidial infection in domestic farm animals such as cattle and sheep are well-known (Angus, 1988; Casey, 1991). Some work has been carried out on levels of infection in deer species (Tzipori and Campbell, 1981; Van Winkle, 1985; Heuschele et al., 1986; Fayer et al., 1996; Majewska et al., 1997; Deng and Cliver, 1999; Rickard et al., 1999; Sturdee et al., 1999), and a limited amount of work has been reported on red deer (Tzipori et al., 1981; Orr et al., 1985; Angus, 1988; Simpson, 1992). The majority of the previously published work on Cryptosporidium in red deer has focused on outbreaks of diarrhoeal disease caused by the parasite infecting young calves. There have been no published investigations of Cryptosporidium infection levels among healthy adult red deer. Deer farming has grown as an industry in Ireland in recent years, and cryptosporidial disease is a recognised problem among the deer herds, causing considerable illness and sometimes death in young deer calves. This work monitored the levels of Cryptosporidium infection in a red deer herd in a non-outbreak situation to see if there was any seasonal fluctuation in numbers of oocysts shed by the deer, which would indicate a particular pattern of infection.
2. Materials and methods 2.1. Sample collection From May 1996 to May 1997, fresh faecal samples were collected on an approximate monthly basis from the pasture of a group of 40 red deer hinds. These deer formed part of a commercially farmed deer herd. Faecal samples were also taken on four occasions from January to May 1997 from a group of 6-month-old deer calves, which were part of the same herd. The calves were extensively managed and were reared in uncrowded outdoor conditions. They received colostrum from their dams in the first hours after birth. On two of the four occasions, samples were taken from the floor of the facility in which the calves were housed, and on the other two occasions, samples were taken from the calves’ pasture. Faeces were deemed to be fresh if they were moist and/or warm with no evidence of drying. In the pasture situation, efforts were made to collect faeces from each quarter of the pasture in order to maximise the possibility of collecting samples from separate individuals. All faecal samples were collected in plastic bags and transported back to the laboratory in a cool box. 5 g of each faecal sample were weighed out and mixed thoroughly with 4% potassium dichromate in order to preserve the samples. The samples were then stored in 25 ml universal containers at 4◦ C until they were processed by water–ether concentration and sucrose density flotation. 2.2. Seeding experiments Faeces of red deer, which had been previously determined to be Cryptosporidium-free following water–ether concentration, sucrose density-flotation and treatment with an FITClabeled monoclonal antibody, were divided into samples of 1 g each. Each sample was seeded with 105 , 104 , 5 × 103 or 103 Cryptosporidium oocysts, suspended in 4% potassium dichro-
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mate and processed by water–ether concentration, sucrose density-flotation and treatment with an FITC-labeled monoclonal antibody. Of the final concentrate of each sample, 10% was subjected to the identification procedure, and the recovery efficiency for each sample was calculated as follows: Recovery efficiency (%) =
(no. of oocysts in final concentrate)(10) × 100 no. of oocysts in seed
2.3. Water–ether concentration The faecal suspension (5 g faeces in 25 ml 4% potassium dichromate) was processed by the water–ether concentration technique of Ferreira et al., 1962. In initial trials of the procedures used for faecal analysis, it was found that the identification of oocysts from faeces that had been treated with the water–ether method alone was extremely difficult. A lot of occluding debris was present together with large numbers of autofluorescent particles. It was decided, therefore, to further purify the faecal sample prior to the identification procedure by incorporating a sucrose density-flotation step. This considerably reduced the debris seen during the identification procedure and made the enumeration of oocysts easier. 2.4. Sucrose density flotation Using a syringe, the final volume of 10 ml in the centrifuge tube was underlain with 10 ml of cold sucrose solution (specific gravity 1.18 at 4◦ C). The tube was centrifuged at 1000 × g for 15 min. The interface and remaining supernatant were drawn off using a pasteur pipette and placed in a 50 ml centrifuge tube. The solution was washed three times in distilled water. The supernatant was decanted and the pellet was resuspended, placed into microfuge tubes and centrifuged for 30 s at 10,000 × g. The resulting pellets were pooled into one tube, which was centrifuged at 10,000 × g for 30 s. The volume in the tube was reduced to 1 ml, the pellet was resuspended and retained for the identification procedure. 2.5. Identification procedure Four 25 l replicates of the final concentrate were suspended on to a multispot microscope slide (Hendley, Essex). Care was taken to ensure that each well was evenly covered. The slide was dried in an incubator set at 37◦ C, after which it was fixed in methanol for 5 min and allowed to air dry. 25 l of a 1:20 dilution of monoclonal antibody (FL-Crypt-a-Glo, Waterborne Inc., New Orleans, USA) were added to each well of the slide, and it was incubated in a humid chamber in the dark at 37◦ C for 30 min. The residual monoclonal antibody was washed off by rinsing the slide in a stream of phosphate buffered saline (PBS, 0.1 M, pH 7.2) from a wash bottle. The slide was washed three times in PBS, three minutes to each wash. The excess PBS was allowed to drain off the slide and the slide was air-dried. 10 l of mounting medium (40:60, 150 mm PBS:Glycerol, 2% Dabco (Sigma)) were placed on each well of the slide and a 22 mm ×50 mm coverslip was applied. The slide was then inverted onto a piece of tissue to allow excess mounting medium to drain off. The
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coverslip was sealed with clear nail varnish. The slide was examined under fluorescence microscopy using an Olympus BL-FLA epifluorescence microscope equipped with a blue filter block for FITC (excitation 490 nm, emission 510 nm). Slides were scanned using the ×20 objective and oocysts were confirmed using the ×40 objective. Oocysts showed apple green fluorescence, a spherical shape and were 4–6 m in diameter.
3. Results 3.1. Seeding experiments Seeding experiments carried out on deer faecal samples to determine the recovery efficiency of the water–ether sedimentation method followed by sucrose flotation showed that, of the four seed sizes used, the 104 oocysts per gram of faeces (opg) seed size recorded the highest mean recovery efficiency (60.3%) (Table 1). The lowest recovery seen was 30.6% for the 103 opg seed size, this seed size also had the smallest range of recoveries. The greatest range in recoveries was seen for the 105 opg seed size, which also recorded the highest overall recovery efficiency of 87.9%. A one-way analysis of variance (ANOVA) carried out to determine the effect of seed size on the recovery efficiency of this method showed that seed size did have a significant effect on the recovery efficiencies obtained (P = 0.0162). 3.2. Number of oocysts excreted by adult hinds Of the 290 faecal samples taken from adult hinds, 114 (39.3%) were positive for Cryptosporidium oocysts. The greatest number of positive samples was seen in June 1996 when 19 of the 30 samples (63.3%) contained Cryptosporidium oocysts (Table 2). The least number of positive samples was seen in May 1996, when none of the samples was positive for oocysts. On no occasion were Cryptosporidium oocysts found in all faecal samples taken in a particular month. The greatest numbers of oocysts seen in individual samples were seen in June 1996 and May 1997, and both these months coincided with the time of calving on the farm. In June 1996, two samples had over 103 opg, and in May 1997 numbers were higher again, with three samples having over 104 opg. In comparison to the other 8 months in which sampling took place, these numbers were very high, given that the maximum number of oocysts seen in any of the other months was 148 opg in January 1997. Table 1 Mean recovery of the water–ether sedimentation method followed by sucrose flotation on 1 g samples of deer faeces seeded with Cryptosporidium parvum oocysts Seed size (opg)
n
Mean ± S.D. (%)
Range (%)
100000 10000 5000 1000
5 5 5 5
47.6 ± 22.6 60.3 ± 11.5 34.6 ± 12.8 30.6 ± 10.5
34.6–87.9 42.4–71.1 24.4–57.0 17.0–41.0
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Table 2 Mean oocyst counts for faecal samples taken from adult hinds from May 1996 to May 1997 Month
n
No. of positive samples
Mean ± S.D. (opg)
Range (opg)
May June July August October November January February April May
30 30 30 30 30 30 30 20 30 30
0 19 16 14 8 17 6 9 12 13
0.0 ± 0.0 321.4 ± 1030.3 2.8 ± 3.3 2.8 ± 3.5 2.1 ± 4.0 3.8 ± 4.4 6.1 ± 27.0 3.9 ± 10.1 1.7 ± 3.0 3796.8 ± 13503.4
0 0–4704 0–12 0–12 0–15 0–15 0–148 0–44 0–12 0–67590
On the whole, mean oocyst counts were low with high standard deviations reflecting the number of negative samples seen in the samples from each month. Large increases in mean oocyst counts were seen in June 1996 (321.4 opg) and May 1997 (3796.8 opg). Again, for these months, the standard deviations were very large, however, which reflects the fact that many negative samples were taken in each of these months and only a small proportion of samples contained high numbers of oocysts. Hence, the greatest ranges in recoveries were recorded for these months. From June to July 1996, there was a marked fall-off in the mean number of oocysts recorded. Mean values fell from 321.4 opg to 2.8 opg. When sampling took place in July, all the hinds had calved. When the 2 months that coincided with calving are excluded, the month that showed the highest mean number of oocysts was January 1997 with a value of 6.1 opg. This value was, however, very much influenced by the presence of one sample, which had 148 opg. 3.3. Numbers of oocysts excreted by calves Twenty one of the 35 faecal samples (60%) taken from calves from January to May 1997 were positive for Cryptosporidium oocysts (Table 3). Of the 4 months in which sampling took place, January was the only one in which all samples were positive. The number of samples taken in that month was, however, lower than that taken in the other 3 months. The highest mean oocyst value for calf faecal samples was recorded in May 1997 (20.8 opg) with three of ten samples having opg values of >25. This peak in numbers of oocysts excreted Table 3 Mean oocyst counts for faecal samples taken from deer calves from January 1997 to May 1997 Month
n
No. of positive samples
Mean ± S.D. (opg)
Range (opg)
Januarya Februarya April May
5 10 10 10
5 4 6 6
4.4 ± 3.3 13.4 ± 30.0 3.0 ± 3.3 20.8 ± 35.3
2–10 0–96 0–8 0–112
a
Calves indoors.
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by calves coincided with a peak in the oocyst opg recorded in adult deer faecal samples. The greatest range in numbers of oocysts in calf faecal samples was seen also in May.
4. Discussion The pattern of oocyst shedding by adult hinds throughout the year was characterised by low levels of oocyst excretion in all months, except for those months in which calving took place (June 1996 and May 1997), when there was an increase in the numbers of oocysts shed in the faeces. Cryptosporidium is widely accepted as being a disease of neonatal ruminants and as such, most of the published literature concentrates on describing the disease state among neonates (Anderson, 1981; Pearson et al., 1982) and on documenting outbreaks of cryptosporidiosis in various animal-rearing facilities such as farms (Angus, 1988), research stations (Tzipori et al., 1981) and zoos (Van Winkle, 1985). On the study farm, only one calf was lost to diarrhoeal illness during the calving season of 1996, and there was no calf mortality during the 1997 calving season. It seems, however, that the adult hinds harboured asymptomatic Cryptosporidium infections all year-round and that oocyst excretion increased at calving time. The factors responsible for this increase are not known. It may perhaps have been precipitated by changes in the hormonal or immune status of the hinds during the calving season or by increases in the hinds’ stress levels at calving time. Asymptomatic shedding of Cryptosporidium oocysts has been reported in cattle. Scott et al. (1995), when analysing faecal samples from two herds of adult beef cattle, found that 62.4% of the samples were positive for Cryptosporidium oocysts. On one farm cryptosporidial diarrhoea among calves had been a problem for years, on the other farm this problem had not been recorded. The percentage of positive samples among adult cattle on each farm was similar. In contrast to the situation seen on the farm used in the present study, a periparturient rise in oocyst excretion was not recorded in either of the cattle herds studied. Lorenzo-Lorenzo et al. (1993) in a study of adult cattle that were not manifesting any symptoms of cryptosporidial infection found that 71.5% of faecal samples taken from the cattle were positive for Cryptosporidium oocysts. Asymptomatic shedding of Cryptosporidium oocysts was not observed among adult horses in an American study. No yearlings or mares were found to be positive for Cryptosporidium infection (Xiao and Herd, 1994). In the course of this work was not possible to repeatedly obtain samples from individual deer because the study farm was a commercial enterprise and not a research facility. Fresh faecal samples were collected from the particular pasture that the group of hinds was grazing, and it cannot be assumed that each sample came from a different animal. It can still be seen, however, that much greater numbers of oocysts were entering the environment during calving time than at any other time of year. This greater level of environmental contamination at calving time and the apparent year-round shedding of oocysts by hinds, could possibly lead to a situation where a pasture as a whole could become contaminated with Cryptosporidium. Endemic contamination of a pasture that was being grazed by red deer was thought to be one of the major factors that contributed to an outbreak of cryptosporidiosis among deer on a Scottish farm. The contamination had occurred when a Cryptosporidium-infected herd of cattle had previously used the same pasture for grazing (Angus, 1988). Cryptosporidia
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isolated from the cattle and from the red deer both infected laboratory animals and were serologically indistinguishable by an indirect immunofluorescence method (Tzipori et al., 1981). On the deer farm used in the present study, excessive contamination of one pasture was not allowed to occur because the deer herd was moved regularly to prevent a build up of contamination and to allow for the regrowth of grass. This management technique may have been a contributing factor to the extremely low level of calf mortality recorded on the farm during the 1996 and 1997 calving seasons. Orr et al. (1985) reported on two outbreaks of Cryptosporidium infection among red deer calves. The first outbreak involved a group of calves that had been captured from the wild and had not received colostrum from their dams. These calves were housed indoors in pens in small groups and were hand reared. At a few weeks of age the calves developed scour and all died within a few days of the onset of illness. The second outbreak involved a group of calves that were assumed to have received colostrum from their dams and were also hand reared indoors. The majority of these calves developed scour and died within 3 days of the onset of illness. Following on from these outbreaks, the workers recommended that good hygiene was essential when calves were being reared indoors and that a feed of colostrum within the first 24 h of life had a protective effect on calves. The fact that calves in the present study were extensively managed, being born and reared in uncrowded outdoor conditions and receiving colostrum from their dams in the first hours of life, may have contributed to the lack of Cryptosporidium-associated disease among calves on the farm during the study period. The calves from the 1996 calving season were housed during the winter months, whereas the hinds remained outside due to the mild weather during the winter of 1996–1997. Faecal samples taken in January and February 1997 from the floor of the calves’ housing facility showed that the calves were excreting low numbers of Cryptosporidium oocysts in their faeces. The potential for transmission of infection in a situation such as that is high, due to the crowded conditions and the difficulty of thoroughly disinfecting the housing area (Orr et al., 1985). The calves did not, however, show any signs of illness and, similarly to the adult hinds, appeared to be asymptomatically shedding oocysts. The faecal samples taken from calves in April and May were taken when the calves had been pastured outside in a separate pasture to that of the adult hinds. The mean opg value for April was again low (3.3 opg), but a peak was seen in May of 20.8 opg. Two of the 10 samples taken in May had 112 and 40 opg, which were very high values for the calf samples in general. Interestingly, this peak in oocysts numbers occurred on the same sampling occasion as the peak recorded in the samples from the hinds. This may perhaps indicate that an increase in oocyst shedding occurs due to seasonal factors and is independent of whether or not an animal is calving. Again, even with these higher opg values, there was no apparent clinical illness among calves. The numbers of oocysts shed by adult hinds and young red deer in the present study were far smaller that the numbers of oocysts generally shed by animals manifesting clinical cryptosporidiosis. For example, bovine calves with severe Cryptosporidium infection can shed 106 –107 opg at the peak of their illness (Henriksen, 1988). Nonetheless, the fact that the deer were asymptomatically shedding oocysts year-round with a periparturient increase is of interest, in that it reveals the presence of Cryptosporidium infection among the deer in a non-outbreak situation.
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Acknowledgements The authors gratefully acknowledge the Science Faculty, Trinity College, Dublin, for the provision of funding that allowed this work to take place; Professor Huw Smith and his colleagues at the Scottish Parasite Diagnostic Laboratory for the valuable training they provided and Jonathan and Betty Sykes for their kindness in allowing us to carry out our work on their deer farm. References Anderson, B.C., 1981. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Am. Vet. Med. Assoc. 178 (9), 982–984. Angus, K.W., 1988. Mammalian cryptosporidiosis: a veterinary perspective. In: Angus, K.W., Blewett, D.A. (Eds), Cryptosporidiosis. Proceedings of the First International Workshop. The Animal Diseases Research Association, pp. 43–55. Casey, M.J., 1991. Cryptosporidium and bovine cryptosporidiosis: a review. Irish Vet. J. 44, 2–7. Current, W.L., Garcia, L.S., 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4, 325–358. Deng, M.Q., Cliver, D.O., 1999. Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals. Parasitol. Res. 85 (8/9), 733–736. Fayer, R., Ungar, B.L.P., 1986. Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev. 50 (4), 458–483. Fayer, R., Fischer, J.R., Sewell, C.T., Kavanaugh, D.M., Osborn, D.A., 1996. Spontaneous cryptosporidiosis in captive white-tailed deer (Odocoileus virginianus). J. Wildlife Dis. 32 (4), 619–622. Ferreira, L.F., Morteo, R.E., Silva, J.R., 1962. Padronizaçã de técnicas para exame parasitológico das fezes. J. Bras. Med. 6, 241–257. Henriksen, S.A., 1988. Epidemiology of cryptosporidiosis in calves. In: Angus, K.W., Blewett, D.A. (Eds.), Cryptosporidiosis. Proceedings of the First International Workshop. The Animal Diseases Research Association, pp. 79–83. Heuschele, W.P., Oosterhius, J., Janssen, D., Rebinson, P.T., Ensley, P.K., Meier, J.E., Olson, T., Anderson, M.P., Benirschke, K., 1986. Cryptosporidial infections in captive wild animals. J. Wildlife Dis. 22, 493–496. Lorenzo-Lorenzo, M.J., Ares-Mazas, E., Villacorta Martinez de Maturana, I., 1993. Detection of oocysts and IgG antibodies to Cryptosporidium parvum in asymptomatic adult cattle. Vet. Parasitol. 47, 9–15. Majewska, A.C., Kasprzak, W., Werner, A., 1997. Prevalence of Cryptosporidium in mammals housed in Poznan Zoological Garden Poland. Acta Parasitol. 42 (4), 195–198. Orr, M.B., Mackintosh, C.G., Suttie, J.M., 1985. Cryptosporidiosis in deer calves. New Zealand Vet. J. 33, 151–152. Pearson, G.R., Logan, E.F., McNulty, M.S., 1982. Distribution of Cryptosporidia within the gastrointestinal tract of young calves. Res. Vet. Sci. 33, 228–231. Rickard, L.G., Siefker, C., Boyle, C.R., Gentz, E.J., 1999. The prevalence of Cryptosporidium and Giardia spp. in fecal samples from free-ranging white-tailed deer (Odocoileus virginianus) in the southeastern United States. J. Vet. Diagnostic Invest. 11 (1), 65–72. Scott, C.A., Smith, H.V., Mtambo, M.M.A., Gibbs, H.A., 1995. An epidemiological study of Cryptosporidium parvum in two herds of adult beef cattle. Vet. Parasitol. 57, 277–288. Simpson, V.R., 1992. Cryptosporidiosis in newborn red deer. Vet. Record 130, 116–118. Sturdee, A.P., Chalmers, R.M., Bull, S.A., 1999. Detection of Cryptosporidium oocysts in wild mammals of mainland Britain. Vet. Parasitol. 80, 273–280. Tzipori, S., Campbell, I., 1981. Prevalence of Cryptosporidium antibodies in 10 animal species. J. Clin. Microbiol. 14 (4), 455–456. Tzipori, S., Angus, K.W., Campbell, I., Sherwood, D., 1981. Diarrhea in young red deer associated with infection with Cryptosporidium. J. Infect. Dis. 144 (2), 170–175. Van Winkle, T.J., 1985. Cryptosporidiosis in young artiodactyls. J. Am. Vet. Assoc. 187 (11), 1170–1172. Xiao, L., Herd, R.P., 1994. Epidemiology of equine Cryptosporidium and Giardia infections. Equine Vet. J. 26 (1), 14–17.
Asymptomatic shedding of Cryptosporidium oocysts by red deer hinds and calves H.E. Skerrett∗ , C.V. Holland Department of Zoology, Trinity College, Dublin 2, Ireland Received 30 May 2000; received in revised form 25 August 2000; accepted 6 September 2000
Abstract Levels of Cryptosporidium infection in a group of red deer were monitored over a period of 1 year. Faecal samples were examined on an approximate monthly basis from adult hinds and calves for the presence of Cryptosporidium oocysts. The water–ether sedimentation method followed by sucrose flotation and a monoclonal antibody identification procedure were used. It was found that apparently healthy adult deer were shedding low numbers of oocysts in their faeces throughout the year and that there appeared to be a periparturient increase in the numbers of oocysts shed. Samples taken from 6-month-old deer calves, both in-house and on pasture, had low numbers of Cryptosporidium oocysts, indicating that the calves were also asymptomatically shedding oocysts. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium spp.; Red deer; Asymptomatic shedding; Faecal shedding
1. Introduction Cryptosporidium parvum is a coccidian, protozoan parasite that has a life cycle similar to that seen in other coccidian parasites. Excystation and an endogenous asexual stage are followed by a sexual stage resulting in the production and discharge of oocysts in the faeces (Current and Garcia, 1991). Cryptosporidium can cause a self-limiting diarrhoeal disease in immunocompetent humans and an extremely severe, life-threatening illness in the immunocompromised (Fayer and Ungar, 1986). Cryptosporidium is also one of the chief causes of diarrhoeal disease among neonatal ruminants (Angus, 1988). ∗ Corresponding author. Present address: Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. Tel.: +353-1-6687988/ext. 2606; fax: +353-1-6608656. E-mail address: [email protected] (H.E. Skerrett).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 4 0 5 - 2
240
H.E. Skerrett, C.V. Holland / Veterinary Parasitology 94 (2001) 239–246
Much work has been carried out on the veterinary disease caused by Cryptosporidium, and patterns of cryptosporidial infection in domestic farm animals such as cattle and sheep are well-known (Angus, 1988; Casey, 1991). Some work has been carried out on levels of infection in deer species (Tzipori and Campbell, 1981; Van Winkle, 1985; Heuschele et al., 1986; Fayer et al., 1996; Majewska et al., 1997; Deng and Cliver, 1999; Rickard et al., 1999; Sturdee et al., 1999), and a limited amount of work has been reported on red deer (Tzipori et al., 1981; Orr et al., 1985; Angus, 1988; Simpson, 1992). The majority of the previously published work on Cryptosporidium in red deer has focused on outbreaks of diarrhoeal disease caused by the parasite infecting young calves. There have been no published investigations of Cryptosporidium infection levels among healthy adult red deer. Deer farming has grown as an industry in Ireland in recent years, and cryptosporidial disease is a recognised problem among the deer herds, causing considerable illness and sometimes death in young deer calves. This work monitored the levels of Cryptosporidium infection in a red deer herd in a non-outbreak situation to see if there was any seasonal fluctuation in numbers of oocysts shed by the deer, which would indicate a particular pattern of infection.
2. Materials and methods 2.1. Sample collection From May 1996 to May 1997, fresh faecal samples were collected on an approximate monthly basis from the pasture of a group of 40 red deer hinds. These deer formed part of a commercially farmed deer herd. Faecal samples were also taken on four occasions from January to May 1997 from a group of 6-month-old deer calves, which were part of the same herd. The calves were extensively managed and were reared in uncrowded outdoor conditions. They received colostrum from their dams in the first hours after birth. On two of the four occasions, samples were taken from the floor of the facility in which the calves were housed, and on the other two occasions, samples were taken from the calves’ pasture. Faeces were deemed to be fresh if they were moist and/or warm with no evidence of drying. In the pasture situation, efforts were made to collect faeces from each quarter of the pasture in order to maximise the possibility of collecting samples from separate individuals. All faecal samples were collected in plastic bags and transported back to the laboratory in a cool box. 5 g of each faecal sample were weighed out and mixed thoroughly with 4% potassium dichromate in order to preserve the samples. The samples were then stored in 25 ml universal containers at 4◦ C until they were processed by water–ether concentration and sucrose density flotation. 2.2. Seeding experiments Faeces of red deer, which had been previously determined to be Cryptosporidium-free following water–ether concentration, sucrose density-flotation and treatment with an FITClabeled monoclonal antibody, were divided into samples of 1 g each. Each sample was seeded with 105 , 104 , 5 × 103 or 103 Cryptosporidium oocysts, suspended in 4% potassium dichro-
H.E. Skerrett, C.V. Holland / Veterinary Parasitology 94 (2001) 239–246
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mate and processed by water–ether concentration, sucrose density-flotation and treatment with an FITC-labeled monoclonal antibody. Of the final concentrate of each sample, 10% was subjected to the identification procedure, and the recovery efficiency for each sample was calculated as follows: Recovery efficiency (%) =
(no. of oocysts in final concentrate)(10) × 100 no. of oocysts in seed
2.3. Water–ether concentration The faecal suspension (5 g faeces in 25 ml 4% potassium dichromate) was processed by the water–ether concentration technique of Ferreira et al., 1962. In initial trials of the procedures used for faecal analysis, it was found that the identification of oocysts from faeces that had been treated with the water–ether method alone was extremely difficult. A lot of occluding debris was present together with large numbers of autofluorescent particles. It was decided, therefore, to further purify the faecal sample prior to the identification procedure by incorporating a sucrose density-flotation step. This considerably reduced the debris seen during the identification procedure and made the enumeration of oocysts easier. 2.4. Sucrose density flotation Using a syringe, the final volume of 10 ml in the centrifuge tube was underlain with 10 ml of cold sucrose solution (specific gravity 1.18 at 4◦ C). The tube was centrifuged at 1000 × g for 15 min. The interface and remaining supernatant were drawn off using a pasteur pipette and placed in a 50 ml centrifuge tube. The solution was washed three times in distilled water. The supernatant was decanted and the pellet was resuspended, placed into microfuge tubes and centrifuged for 30 s at 10,000 × g. The resulting pellets were pooled into one tube, which was centrifuged at 10,000 × g for 30 s. The volume in the tube was reduced to 1 ml, the pellet was resuspended and retained for the identification procedure. 2.5. Identification procedure Four 25 l replicates of the final concentrate were suspended on to a multispot microscope slide (Hendley, Essex). Care was taken to ensure that each well was evenly covered. The slide was dried in an incubator set at 37◦ C, after which it was fixed in methanol for 5 min and allowed to air dry. 25 l of a 1:20 dilution of monoclonal antibody (FL-Crypt-a-Glo, Waterborne Inc., New Orleans, USA) were added to each well of the slide, and it was incubated in a humid chamber in the dark at 37◦ C for 30 min. The residual monoclonal antibody was washed off by rinsing the slide in a stream of phosphate buffered saline (PBS, 0.1 M, pH 7.2) from a wash bottle. The slide was washed three times in PBS, three minutes to each wash. The excess PBS was allowed to drain off the slide and the slide was air-dried. 10 l of mounting medium (40:60, 150 mm PBS:Glycerol, 2% Dabco (Sigma)) were placed on each well of the slide and a 22 mm ×50 mm coverslip was applied. The slide was then inverted onto a piece of tissue to allow excess mounting medium to drain off. The
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coverslip was sealed with clear nail varnish. The slide was examined under fluorescence microscopy using an Olympus BL-FLA epifluorescence microscope equipped with a blue filter block for FITC (excitation 490 nm, emission 510 nm). Slides were scanned using the ×20 objective and oocysts were confirmed using the ×40 objective. Oocysts showed apple green fluorescence, a spherical shape and were 4–6 m in diameter.
3. Results 3.1. Seeding experiments Seeding experiments carried out on deer faecal samples to determine the recovery efficiency of the water–ether sedimentation method followed by sucrose flotation showed that, of the four seed sizes used, the 104 oocysts per gram of faeces (opg) seed size recorded the highest mean recovery efficiency (60.3%) (Table 1). The lowest recovery seen was 30.6% for the 103 opg seed size, this seed size also had the smallest range of recoveries. The greatest range in recoveries was seen for the 105 opg seed size, which also recorded the highest overall recovery efficiency of 87.9%. A one-way analysis of variance (ANOVA) carried out to determine the effect of seed size on the recovery efficiency of this method showed that seed size did have a significant effect on the recovery efficiencies obtained (P = 0.0162). 3.2. Number of oocysts excreted by adult hinds Of the 290 faecal samples taken from adult hinds, 114 (39.3%) were positive for Cryptosporidium oocysts. The greatest number of positive samples was seen in June 1996 when 19 of the 30 samples (63.3%) contained Cryptosporidium oocysts (Table 2). The least number of positive samples was seen in May 1996, when none of the samples was positive for oocysts. On no occasion were Cryptosporidium oocysts found in all faecal samples taken in a particular month. The greatest numbers of oocysts seen in individual samples were seen in June 1996 and May 1997, and both these months coincided with the time of calving on the farm. In June 1996, two samples had over 103 opg, and in May 1997 numbers were higher again, with three samples having over 104 opg. In comparison to the other 8 months in which sampling took place, these numbers were very high, given that the maximum number of oocysts seen in any of the other months was 148 opg in January 1997. Table 1 Mean recovery of the water–ether sedimentation method followed by sucrose flotation on 1 g samples of deer faeces seeded with Cryptosporidium parvum oocysts Seed size (opg)
n
Mean ± S.D. (%)
Range (%)
100000 10000 5000 1000
5 5 5 5
47.6 ± 22.6 60.3 ± 11.5 34.6 ± 12.8 30.6 ± 10.5
34.6–87.9 42.4–71.1 24.4–57.0 17.0–41.0
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Table 2 Mean oocyst counts for faecal samples taken from adult hinds from May 1996 to May 1997 Month
n
No. of positive samples
Mean ± S.D. (opg)
Range (opg)
May June July August October November January February April May
30 30 30 30 30 30 30 20 30 30
0 19 16 14 8 17 6 9 12 13
0.0 ± 0.0 321.4 ± 1030.3 2.8 ± 3.3 2.8 ± 3.5 2.1 ± 4.0 3.8 ± 4.4 6.1 ± 27.0 3.9 ± 10.1 1.7 ± 3.0 3796.8 ± 13503.4
0 0–4704 0–12 0–12 0–15 0–15 0–148 0–44 0–12 0–67590
On the whole, mean oocyst counts were low with high standard deviations reflecting the number of negative samples seen in the samples from each month. Large increases in mean oocyst counts were seen in June 1996 (321.4 opg) and May 1997 (3796.8 opg). Again, for these months, the standard deviations were very large, however, which reflects the fact that many negative samples were taken in each of these months and only a small proportion of samples contained high numbers of oocysts. Hence, the greatest ranges in recoveries were recorded for these months. From June to July 1996, there was a marked fall-off in the mean number of oocysts recorded. Mean values fell from 321.4 opg to 2.8 opg. When sampling took place in July, all the hinds had calved. When the 2 months that coincided with calving are excluded, the month that showed the highest mean number of oocysts was January 1997 with a value of 6.1 opg. This value was, however, very much influenced by the presence of one sample, which had 148 opg. 3.3. Numbers of oocysts excreted by calves Twenty one of the 35 faecal samples (60%) taken from calves from January to May 1997 were positive for Cryptosporidium oocysts (Table 3). Of the 4 months in which sampling took place, January was the only one in which all samples were positive. The number of samples taken in that month was, however, lower than that taken in the other 3 months. The highest mean oocyst value for calf faecal samples was recorded in May 1997 (20.8 opg) with three of ten samples having opg values of >25. This peak in numbers of oocysts excreted Table 3 Mean oocyst counts for faecal samples taken from deer calves from January 1997 to May 1997 Month
n
No. of positive samples
Mean ± S.D. (opg)
Range (opg)
Januarya Februarya April May
5 10 10 10
5 4 6 6
4.4 ± 3.3 13.4 ± 30.0 3.0 ± 3.3 20.8 ± 35.3
2–10 0–96 0–8 0–112
a
Calves indoors.
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by calves coincided with a peak in the oocyst opg recorded in adult deer faecal samples. The greatest range in numbers of oocysts in calf faecal samples was seen also in May.
4. Discussion The pattern of oocyst shedding by adult hinds throughout the year was characterised by low levels of oocyst excretion in all months, except for those months in which calving took place (June 1996 and May 1997), when there was an increase in the numbers of oocysts shed in the faeces. Cryptosporidium is widely accepted as being a disease of neonatal ruminants and as such, most of the published literature concentrates on describing the disease state among neonates (Anderson, 1981; Pearson et al., 1982) and on documenting outbreaks of cryptosporidiosis in various animal-rearing facilities such as farms (Angus, 1988), research stations (Tzipori et al., 1981) and zoos (Van Winkle, 1985). On the study farm, only one calf was lost to diarrhoeal illness during the calving season of 1996, and there was no calf mortality during the 1997 calving season. It seems, however, that the adult hinds harboured asymptomatic Cryptosporidium infections all year-round and that oocyst excretion increased at calving time. The factors responsible for this increase are not known. It may perhaps have been precipitated by changes in the hormonal or immune status of the hinds during the calving season or by increases in the hinds’ stress levels at calving time. Asymptomatic shedding of Cryptosporidium oocysts has been reported in cattle. Scott et al. (1995), when analysing faecal samples from two herds of adult beef cattle, found that 62.4% of the samples were positive for Cryptosporidium oocysts. On one farm cryptosporidial diarrhoea among calves had been a problem for years, on the other farm this problem had not been recorded. The percentage of positive samples among adult cattle on each farm was similar. In contrast to the situation seen on the farm used in the present study, a periparturient rise in oocyst excretion was not recorded in either of the cattle herds studied. Lorenzo-Lorenzo et al. (1993) in a study of adult cattle that were not manifesting any symptoms of cryptosporidial infection found that 71.5% of faecal samples taken from the cattle were positive for Cryptosporidium oocysts. Asymptomatic shedding of Cryptosporidium oocysts was not observed among adult horses in an American study. No yearlings or mares were found to be positive for Cryptosporidium infection (Xiao and Herd, 1994). In the course of this work was not possible to repeatedly obtain samples from individual deer because the study farm was a commercial enterprise and not a research facility. Fresh faecal samples were collected from the particular pasture that the group of hinds was grazing, and it cannot be assumed that each sample came from a different animal. It can still be seen, however, that much greater numbers of oocysts were entering the environment during calving time than at any other time of year. This greater level of environmental contamination at calving time and the apparent year-round shedding of oocysts by hinds, could possibly lead to a situation where a pasture as a whole could become contaminated with Cryptosporidium. Endemic contamination of a pasture that was being grazed by red deer was thought to be one of the major factors that contributed to an outbreak of cryptosporidiosis among deer on a Scottish farm. The contamination had occurred when a Cryptosporidium-infected herd of cattle had previously used the same pasture for grazing (Angus, 1988). Cryptosporidia
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isolated from the cattle and from the red deer both infected laboratory animals and were serologically indistinguishable by an indirect immunofluorescence method (Tzipori et al., 1981). On the deer farm used in the present study, excessive contamination of one pasture was not allowed to occur because the deer herd was moved regularly to prevent a build up of contamination and to allow for the regrowth of grass. This management technique may have been a contributing factor to the extremely low level of calf mortality recorded on the farm during the 1996 and 1997 calving seasons. Orr et al. (1985) reported on two outbreaks of Cryptosporidium infection among red deer calves. The first outbreak involved a group of calves that had been captured from the wild and had not received colostrum from their dams. These calves were housed indoors in pens in small groups and were hand reared. At a few weeks of age the calves developed scour and all died within a few days of the onset of illness. The second outbreak involved a group of calves that were assumed to have received colostrum from their dams and were also hand reared indoors. The majority of these calves developed scour and died within 3 days of the onset of illness. Following on from these outbreaks, the workers recommended that good hygiene was essential when calves were being reared indoors and that a feed of colostrum within the first 24 h of life had a protective effect on calves. The fact that calves in the present study were extensively managed, being born and reared in uncrowded outdoor conditions and receiving colostrum from their dams in the first hours of life, may have contributed to the lack of Cryptosporidium-associated disease among calves on the farm during the study period. The calves from the 1996 calving season were housed during the winter months, whereas the hinds remained outside due to the mild weather during the winter of 1996–1997. Faecal samples taken in January and February 1997 from the floor of the calves’ housing facility showed that the calves were excreting low numbers of Cryptosporidium oocysts in their faeces. The potential for transmission of infection in a situation such as that is high, due to the crowded conditions and the difficulty of thoroughly disinfecting the housing area (Orr et al., 1985). The calves did not, however, show any signs of illness and, similarly to the adult hinds, appeared to be asymptomatically shedding oocysts. The faecal samples taken from calves in April and May were taken when the calves had been pastured outside in a separate pasture to that of the adult hinds. The mean opg value for April was again low (3.3 opg), but a peak was seen in May of 20.8 opg. Two of the 10 samples taken in May had 112 and 40 opg, which were very high values for the calf samples in general. Interestingly, this peak in oocysts numbers occurred on the same sampling occasion as the peak recorded in the samples from the hinds. This may perhaps indicate that an increase in oocyst shedding occurs due to seasonal factors and is independent of whether or not an animal is calving. Again, even with these higher opg values, there was no apparent clinical illness among calves. The numbers of oocysts shed by adult hinds and young red deer in the present study were far smaller that the numbers of oocysts generally shed by animals manifesting clinical cryptosporidiosis. For example, bovine calves with severe Cryptosporidium infection can shed 106 –107 opg at the peak of their illness (Henriksen, 1988). Nonetheless, the fact that the deer were asymptomatically shedding oocysts year-round with a periparturient increase is of interest, in that it reveals the presence of Cryptosporidium infection among the deer in a non-outbreak situation.
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Acknowledgements The authors gratefully acknowledge the Science Faculty, Trinity College, Dublin, for the provision of funding that allowed this work to take place; Professor Huw Smith and his colleagues at the Scottish Parasite Diagnostic Laboratory for the valuable training they provided and Jonathan and Betty Sykes for their kindness in allowing us to carry out our work on their deer farm. References Anderson, B.C., 1981. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Am. Vet. Med. Assoc. 178 (9), 982–984. Angus, K.W., 1988. Mammalian cryptosporidiosis: a veterinary perspective. In: Angus, K.W., Blewett, D.A. (Eds), Cryptosporidiosis. Proceedings of the First International Workshop. The Animal Diseases Research Association, pp. 43–55. Casey, M.J., 1991. Cryptosporidium and bovine cryptosporidiosis: a review. Irish Vet. J. 44, 2–7. Current, W.L., Garcia, L.S., 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4, 325–358. Deng, M.Q., Cliver, D.O., 1999. Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals. Parasitol. Res. 85 (8/9), 733–736. Fayer, R., Ungar, B.L.P., 1986. Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev. 50 (4), 458–483. Fayer, R., Fischer, J.R., Sewell, C.T., Kavanaugh, D.M., Osborn, D.A., 1996. Spontaneous cryptosporidiosis in captive white-tailed deer (Odocoileus virginianus). J. Wildlife Dis. 32 (4), 619–622. Ferreira, L.F., Morteo, R.E., Silva, J.R., 1962. Padronizaçã de técnicas para exame parasitológico das fezes. J. Bras. Med. 6, 241–257. Henriksen, S.A., 1988. Epidemiology of cryptosporidiosis in calves. In: Angus, K.W., Blewett, D.A. (Eds.), Cryptosporidiosis. Proceedings of the First International Workshop. The Animal Diseases Research Association, pp. 79–83. Heuschele, W.P., Oosterhius, J., Janssen, D., Rebinson, P.T., Ensley, P.K., Meier, J.E., Olson, T., Anderson, M.P., Benirschke, K., 1986. Cryptosporidial infections in captive wild animals. J. Wildlife Dis. 22, 493–496. Lorenzo-Lorenzo, M.J., Ares-Mazas, E., Villacorta Martinez de Maturana, I., 1993. Detection of oocysts and IgG antibodies to Cryptosporidium parvum in asymptomatic adult cattle. Vet. Parasitol. 47, 9–15. Majewska, A.C., Kasprzak, W., Werner, A., 1997. Prevalence of Cryptosporidium in mammals housed in Poznan Zoological Garden Poland. Acta Parasitol. 42 (4), 195–198. Orr, M.B., Mackintosh, C.G., Suttie, J.M., 1985. Cryptosporidiosis in deer calves. New Zealand Vet. J. 33, 151–152. Pearson, G.R., Logan, E.F., McNulty, M.S., 1982. Distribution of Cryptosporidia within the gastrointestinal tract of young calves. Res. Vet. Sci. 33, 228–231. Rickard, L.G., Siefker, C., Boyle, C.R., Gentz, E.J., 1999. The prevalence of Cryptosporidium and Giardia spp. in fecal samples from free-ranging white-tailed deer (Odocoileus virginianus) in the southeastern United States. J. Vet. Diagnostic Invest. 11 (1), 65–72. Scott, C.A., Smith, H.V., Mtambo, M.M.A., Gibbs, H.A., 1995. An epidemiological study of Cryptosporidium parvum in two herds of adult beef cattle. Vet. Parasitol. 57, 277–288. Simpson, V.R., 1992. Cryptosporidiosis in newborn red deer. Vet. Record 130, 116–118. Sturdee, A.P., Chalmers, R.M., Bull, S.A., 1999. Detection of Cryptosporidium oocysts in wild mammals of mainland Britain. Vet. Parasitol. 80, 273–280. Tzipori, S., Campbell, I., 1981. Prevalence of Cryptosporidium antibodies in 10 animal species. J. Clin. Microbiol. 14 (4), 455–456. Tzipori, S., Angus, K.W., Campbell, I., Sherwood, D., 1981. Diarrhea in young red deer associated with infection with Cryptosporidium. J. Infect. Dis. 144 (2), 170–175. Van Winkle, T.J., 1985. Cryptosporidiosis in young artiodactyls. J. Am. Vet. Assoc. 187 (11), 1170–1172. Xiao, L., Herd, R.P., 1994. Epidemiology of equine Cryptosporidium and Giardia infections. Equine Vet. J. 26 (1), 14–17.