Infection patterns of Cryptosporidium and Giardia in calves

v e t e r i n ary

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parasitology

Veterinary Parasitology 55 (1994) 257-262

Short communication

Infection patterns of Cryptosporidium and Giardia in calves Lihua Xiao*, R.P. Herd Department of Veterinary PreventiveMedicine, The Ohio State University College of Veterinary Medicine, 1900 Coffey Road, Columbus, OH 43210, USA Accepted 15 December 1993

Abstract

Faecal samples were collected weekly over a 3 month period from 0- to 20-week-old calves on an Ohio dairy farm, and examined for Cryptosporidium oocysts and Giardia cysts by a quantitative direct immunofluorescence assay. Oocysts and cysts were detected as early as 4 days of age. Shedding of Cryptosporidium oocysts peaked at 1 week and was low by 3 weeks. Some calves, however, continued to pass low numbers of oocysts. Shedding of Giardia cysts peaked at 2 weeks of age with high levels maintained until 7 weeks of age. Most calves continued to pass low numbers of Giardia cysts after weaning. Cumulative infection rates for both Cryptosporidium parvum and Giardia sp. were 100%.

Keywords: Giardia sp.; Cryptosporidium parvum; Cattle-Protozoa; Epidemiology-Protozoa

1. Introduction The high prevalence of Cryptosporidium infection in calves is well known (Angus, 1990). Recent studies indicate that Giardia infection is also widespread in calves (Fischer, 1983; Gasser et al., 1987; TamineUi and Eckert, 1989; Buret et al., 1990; Xiao et al., 1993). Despite the wide prevalence of these two parasites and possible pathogenic roles, quantitative studies on infection patterns are very few (Stein et al., 1983). Most studies on Cryptosporidium infection patterns in calves are qualitative descriptions of the onset, peak and waning of infection (Anderson, 1981; Pavia* Corresponding author. Present address: Immunology Branch, Mail Stop F- 12, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. 0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

SSDI 0304-4017 (93 )00645-F

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sek, 1982; Henriksen and Krogh, 1985; Schulz, 1986; Blewett, 1989). Blewett (1989) studied many quantitative aspects of Cryptosporidium infection in calves, but gave no oocyst shedding patterns of naturally infected animals. The only quantitative study on the oocyst shedding pattern of naturally infected calves was done by Stein et al. ( 1983 ), using a semiquantitative acid-fast staining technique. Few studies have examined Giardia infection patterns in calves. Deshpande and Shastri ( 1981 ), Fischer ( 1983 ) and Nikitin et al. ( 1991 ) described qualitatively the onset of Giardia infection in calves of early ages, while Gasser et al. (1987) studied patterns of Giardia cyst release for 62 days in nine calves. The duration of these studies, however, was too short, because calves can be positive from 11 to 164 days of age (Xiao et al., 1993). The paucity of quantitative studies on these two parasites is partially because of the lack of simple and sensitive quantitative detection methods. The haemacytometer method and acid-fast staining method used by earlier researchers (Stein et al., 1983; Blewett, 1989) in Cryptosporidium detection have low sensitivities and are unable to detect light infections (Arrowood and Sterling, 1989; Cozon et al., 1992; Mtambo et al., 1992). The sucrose gradient flotation method used by Gasser et al. (1987) in Giardia detection also cannot be used in neonatal calves because of the presence of excess fat in faeces. Recently, a quantitative direct immunofluorescence assay (FA) for the simultaneous detection of Cryptosporidium and Giardia infections was developed (Xiao and Herd, 1993 ), which is more sensitive than conventional methods (acid-fast staining, sucrose gradient flotation and zinc sulphate flotation) and can be used in young calves. It was used to determine infection patterns of Cryptosporidium and Giardia in calves in the present study. 2. Materials and methods

2.1. Animals Holstein and Jersey calves on a central Ohio farm known to have a high prevalence of Cryptosporidium and Giardia infections (Xiao et al., 1993 ) were separated from dams shortly after birth and raised in individual hutches before weaning at 7 weeks of age. Animals were chained to hutches, but the chain was long enough to allow contact with calves in neighbouring hutches. At weaning, calves were transferred to one of two pens attached to small exercise yards. Commercial milk replacer was the maj or diet before weaning, replaced by concentrate and hay at weaning. Water was available ad libitum. Most calves had one or more episodes of diarrhoea before weaning and three animals died as the result of diarrhoea.

2.2. Samples Rectal faecal samples were taken from 29 calves younger than 20 weeks of age once every week from the beginning of January to the end of March 1993. Eight-

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een calves born during the period were also sampled. Sampling stopped when calves were older than 20 weeks. Ages of calves at sampling were rounded down by full weeks, thus calves 6 days old were considered as 0 week in age. Five to 22 calves were sampled each week of age, and a total of 274 samples were taken. Numbers of samples taken at each week of age are shown in Table 1.

2.3. Quantitative immunofluorescence assay

Cryptosporidium oocysts and Giardia cysts per gram of faeces were counted by the FA using a commercial kit from Meridian Diagnostics (Cincinnati, OH, USA ) (Xiao and Herd, 1993 ). This FA employs two monoclonal antibodies and is designed for simultaneous detection of Cryptosporidium oocysts and Giardia cysts. The theoretical sensitivity of this test is 100 cysts or 100 oocysts per gram of faeces. Mean cysts or oocysts per gram at each week of age were calculated. Table 1 Infection patterns of Age (weeks)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 !6 17 18 19 20

Cryptosporidium and Giardia in calves

No. sampled

18 15 22 21 19 20 12 12 11 13 10 10 11 12 13 9 11 9 11 10 5

a Oocysts per gram of faeces. b Cysts per gram of faeces.

Cryptosporidium

Giardia

No. positive

Mean ± SD a

No. positive

Mean ± S D b

2 14 21 15 5 6 2 0 0 0 0 0 2 1 1 0 1 1 1 1 1

39± 114 6959440± 8679635 1609850± 2955030 12695±52138 79± 162 100± 275 2525 ± 8684 0 0 0 0 0 45 ± 104 42± 144 9 ± 30 0 27± 90 11 ±33 9 ± 30 40± 126 20± 45

2 8 12 17 16 19 12 10 10 1l 10 8 7 6 6 8 4 5 5 7 4

7±51 6000 ±16166 320982 ± 952600 112610± 193481 103884± 142173 113070± 170073 66500± 70258 11975 ±14710 34991 ± 95004 1146± 1491 5950± 13397 320± 329 33400± 91839 808± 1752 14227 ±46657 8878±22350 7464±24390 956± 1615 8082 ± 26407 710± 1560 2700 ± 5161

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3. Results

Mean Cryptosporidium oocyst counts per gram of faeces were low in calves before 1 week of age (Table 1 ). Oocyst counts peaked at 1 week, and then decreased to low numbers by 3 weeks. The earliest shedding occurred at 4 days of age. Shedding of oocysts continued sporadically with calves as old as 20 weeks passing Cryptosporidium oocysts, but at much lower rates and intensities. All animals sampled sequentially from birth had at least one shedding of Cryptosporidium oocysts. Individual oocyst counts were as high as 26 million oocysts per gram of faeces. Oocysts in this study were morphologically similar to those of Cryptosporidium parvum by both FA and regular microscopic examinations. Mean Giardia cyst counts per gram of faeces in calves were very low before 1 week of age and reached a peak at 2 weeks (Table 1 ). The earliest shedding of Giardia cysts occurred at 4 days of age. Cysts per gram were maintained at high levels until 7 weeks of age. Shedding of Giardia cysts continued long after weaning, but showed a trend of gradual reduction in shedding rates and intensities. All calves sampled sequentially from birth had at least one shedding of Giardia cysts. Individual counts of calves were as high as 4.2 million cysts per gram of faeces. Cysts found in this study were morphologically similar to those of the Giardia duodenalis group by differential interference contrast microscopy. Of the 274 samples taken, 48 (18%) had both Cryptosporidium oocysts and Giardia cysts. Forty-three (90%) of these double positive samples were from calves 1-6 weeks of age.

4. Discussion

Results from this study confirmed findings by earlier researchers that Cryptosporidium infection in calves is mostly a neonatal problem (Anderson, 1981; Pavlasek, 1982; Stein et al., 1983; Henriksen and Krogh, 1985; Schulz, 1986; Blewett, 1989). Release of oocysts started during the first week of life, peaked and decreased quickly, and by 3 weeks of age the infection intensity was very low. Time of first oocyst shedding (4 days) and cumulative infection rate (100%) in this study were similar to those reported by previous workers (Anderson, 1981; Stein et al., 1983; Schulz, 1986; Blewett, 1989). Infection intensities of calves in this report was similar to those reported by Blewett ( 1989 ) in experimentally or naturally infected calves, but lower than those reported by Stein et al. (1983 ) and Schulz (1986), who used semiquantitative detection methods. Reinfection apparently did not change the oocyst shedding pattern significantly, since the pattern seen here was similar to the pattern seen in experimentally infected calves (Blewett, 1989) and lambs (Hill, 1989). Shedding of Cryptosporidium parvum oocysts occurred periodically in calves older than 4 weeks, even long after weaning. This is in agreement with results obtained in a previous study (Xiao et al., 1993 ), where some calves as old as 5.5 months passed oocysts. Henriksen and Krogh (1985) also found Cryptospori-

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dium infection at postmortem in calves older than 2 months and in some adult cattle, with low infection rates and intensities. In contrast, Villacorta et al. ( 1991 ) and Lorenzo Lorenzo et at. ( 1993 ) showed high infection rates and intensities of Cryptosporidium infection in adult cattle. Results of these Spanish studies should be interpreted with caution as they used acid-fast staining, a detection method of low (52%) specificity (Arrowood and Sterling, 1989). In one report 71.75% of asymptomatic adult cattle had four to eight Cryptosporidium oocysts per field at × 400 magnification (Lorenzo Lorenzo et al., 1993 ), an infection of moderate to severe intensities (Henriksen and Krogh, 1985; Arrowood and Sterling, 1989; Xiao et al., 1993 ). This infection rate was even higher than the seroprevalence of the same cattle population in their studies. The infection pattern of Giardia in this study was different from Cryptosporidium. Although the earliest shedding of Giardia cysts and Cryptosporidium oocysts was seen at 4 days of age, infection rates (percent of positives) and intensities (cysts per gram ) of Giardia increased more slowly than those of Cryptosporidium. Giardia infection peaked later and lasted longer than Cryptosporidium. Most animals were still passing Giardia cysts when the study ended, but only a few calves were excreting Cryptosporidium oocysts. Shedding of Giardia cysts was more intensive than the shedding of Cryptosporidium oocysts after 3 weeks of age. The prepatent period of 4 days for Giardia in this study is shorter than that previously reported in experimentally or naturally infected calves (Fischer, 1983; Taminelli et al., 1989). A prepatent period of 7-8 days was found by Taminelli et al. (1989 ) in calves experimentally infected with 1.5-5.1 × 106 Giardia cysts. Fischer (1983) showed that the shedding of Giardia cysts in naturally infected calves began at 2 weeks of age. Giardia infection intensities in this study are much higher than those reported by Gasser et at. (1987 ), where calves were examined after they were 7-8 weeks old. The highest cyst counts occurred at the beginning of their study. As shown in the present study, Giardia infection in calves is most intensive before 7 weeks of age. In the study by Fischer ( 1983 ), the highest infection rates and intensities of Giardia occurred in calves of 5-6 weeks of age, later than those in our study.The role of Cryptosporidium and Giardia infections in the occurrence of diarrhoea on this farm was not clear. Diarrhoea and deaths occurred mainly during the period when calves were in hutches. The highest infection rate and intensity of both Cryptosporidium and Giardia also occurred in this period. Other enteropathogens such as rotavirus and Salmonella, however, were also present on the farm. It is likely that diarrhoea was the result of concurrent infection with multiple pathogens. Most previous investigations on the aetiology of calf diarrhoea did not examine the role of Giardia infection (Moon et al., 1978; Tzipori, 1981; Reynolds et al., 1986; Snodgrass et al., 1986 ). Results from this and previous studies (Xiao et al., 1993 ) suggest that Giardia and Cryptosporidium infections may also be an important cause of diarrhoea. References Anderson, B.C., 1981. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Am. Vet. Med. Assoc., 178: 982-984.

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