Experimental Eimeria alabamensis infection in calves

veterinary parasitology Veterinary Parasitology 53 (1994) 23-32

ELSEVIER

Experimental Eimeria alabamensis infection in calves P. H o o s h m a n d - R a d a'c, C. S v e n s s o n *'b, A. U g g l a a aDepartment of Parasitology, National Veterinary Institute and Swedish University of Agricultural Sciences, P.O. Box 7073, S-750 07 Uppsala, Sweden bExperimental Station, Veterinary lnstitute, P.O. Box 234, S-532 23 Skara, Sweden CDepartment of Cattle and Sheep Diseases, Swedish University of Agricultural Sciences, P.O. Box 7019, S- 750 07 Uppsala, Sweden

(Accepted 20 July 1993)

Abstract Four groups of three conventionally reared 2-month-old bull calves were inoculated with 10 million to 400 million sporulated oocysts of Eimeria alabamensis isolated from Swedish calves which had diarrhoea while at pasture. Their appetite, clinical condition, growth rate and the dry matter content of their faeces were compared with those of three similar but uninoculated calves. The prepatent period was 6-8 days, and the period during which large numbers of oocysts were excreted was 2-7 days. In two of the inoculated calves only a slight softening of the faeces was observed. The other ten calves developed watery diarrhoea, had a poor appetite and appeared depressed. The clinical signs were most severe in the calves which received the highest doses and included signs of abdominal pain and a reluctance to rise. The growth rates of the infected calves were significantly reduced for 18 days after inoculation, and 71 days after inoculation they had not compensated for this period of reduced growth rate. Key words: Eimeria alabamensis; Cattle-Protozoa

1. Introduction The diarrhoea a n d loss o f weight which often affect calves in Sweden when they are first turned out to graze have traditionally been attributed to the sudden change *Corresponding author: Tel. (46)-511-30000; Fax. (46)-511-30134. 0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0304-4017 (93) 00575-J

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in diet. However, more recently it has been suggested that Eimeria species may play a role, mainly because it is commonly found that diarrhoeic calves are excreting large numbers of oocysts of Eimeria alabamensis (Svensson et al., 1993 ). Eimeria alabamensis was first described in the USA by Christensen ( 1941 ). This parasite infects the nucleus of the epithelial cells lining the small intestine (Davis et al., 1957 ) and has been shown to induce diarrhoea in animals infected experimentally with large numbers of oocysts (Davis et al., 1955; Soekardono, 1972; Graubmann, 1986). However, in spite of its worldwide distribution, coccidiosis due to a natural infection with this species has been reported only from northeastern Germany (Gr~ifner et al., 1982 ). This paper describes the results of experiments designed to investigate the pathogenicity of a Swedish isolate of E. alabamensis to calves reared under traditional husbandry conditions.

2. Materials and methods

2.1. Inoculum The oocysts were derived from calves which had diarrhoea while they were at pasture and which had no Eimeria other than E. alabamensis in their faeces. The identification of the species was confirmed by the Central Veterinary Laboratory, Weybridge, UK. The oocysts were maintained in a solution of 2% potassium dichromate at 4 ° C. Before the experiments began, a fresh stock of the oocysts was obtained by propagating them through two 6-week-old calves which were each inoculated with 10 million oocysts. The oocysts were collected at the peak of excretion 8 and 9 days after inoculation and sporulated by standard methods (Anonymous, 1986 ). Shortly before the oocysts were administered to the experimental calves, the potassium dichromate was removed by repeated centrifugation in distilled water. The oocysts were resuspended in distilled water and the number of oocysts per millilitre was counted using a haemocytometer. A measured quantity of inoculum calibrated to contain the number of oocysts required was mixed with about 200 g of concentrates and fed to the calves in feed buckets. To ensure that all the oocysts were ingested, no concentrates had been fed to the calves during the previous 24 h.

2.2. Experimental animals and design Fifteen 2-month-old Swedish Red and White bull calves were used for the experiments. The calves had been purchased when they were 1-2 weeks old from a farm which had no history of clinical coccidiosis. They were housed in individual pens with concrete floors bedded with wood shavings, in a barn isolated from other animals. The calves were fed milk replacer, hay and concentrates, and

P. Hooshmand-Rad et al. / Veterinary Parasitology 53 (1994) 23-32

25

weaned at about 7 weeks of age, approximately 2 weeks before the experiments began. Staff attending the animals changed their shoes and put on protective clothing before entering the barn, in order to minimise the risk of introducing infectious agents. An examination of faecal samples which were taken regularly before the experiments began, revealed that all the calves had low-grade infections with Eimeria species; in four animals, up to 1000 oocysts ofE. alabamensis per gram of faeces were present. The calves were allocated at random to five groups of three animals each. The calves in three of the groups were each inoculated with a single dose of 10 million, 100 million or 400 million sporulated oocysts of E. alabamensis; those in the fourth group each received a trickle infection of 20 million sporulated oocysts per day for 5 days, and the calves in the last group were used as uninoculated controis. All calves were monitored clinically and parasitologically for 28 days. Two of the calves given a single dose of 10 million oocysts and two of the calves dosed with 400 million oocysts were reinoculated with 100 million sporulated oocysts of a homologous strain of E. alabamensis 72 days after their first dose and were monitored for 15 days.

2.3. Faecal examinations Faecal samples were collected daily from the rectum of the calves. The numbers of oocysts were counted by a modified McMaster method (Anonymous, 1986) and the faecal dry matter was measured by the method described by Svensson et al. (1993).

2.4. Clinical condition Each day the calves were examined clinically and their rectal temperature was recorded. Their consumption of concentrates was measured as an indication of appetite. The animals were weighed 5 days before inoculation, on the day of inoculation (Day 0) and 5, 18, 28 and 71 days after inoculation. The four calves which were challenged 72 days after the initial inoculation were also weighed 16 days after the reinoculation (Day 88 ).

2.5. Statistical analyses The dry matter contents of the faeces of the different groups of calves and their weight gains were compared using Student's t-test (Altman, 1991 ), using StatView (Abacus Concepts, Berkeley, CA).

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3. Results

3.1. Initial inoculation Parasitological findings The infected calves began to excrete large numbers of oocysts ofE. alabamensis on Day 8 (Table 1 ). In ten of the calves, more than 1 million oocysts g-1 faeces (OPG) were found for 1-3 days (mean 1.9 days) and more than 5000 OPG for 2-7 days (mean 5.1 days). One of the calves inoculated with 100 million oocysts (Calf 5 ) and one of those inoculated with five doses of 20 million oocysts (Calf 9 ) excreted relatively few oocysts and their peak values were only 100 000 OPG and 70 000 OPG, respectively. The calves which received 400 million oocysts or five doses of 20 million oocysts excreted large numbers of oocysts for longer than the calves in the other groups. However, the largest values of OPG were found in calves inoculated with 10 million or 100 million oocysts. In the uninfected calves, only small increases in the excretion of oocysts were observed.

Changes in clinical condition The increases in the excretion of oocysts by the inoculated calves were preceded by a decrease in the dry matter content of the faeces (Fig. 1 ), which in two of the calves (Calves 5 and 9) was manifested as a softening of the faeces and in the other ten calves was observed clinically as watery diarrhoea. Diarrhoea was first recorded 3-7 days after inoculation and lasted for 1-7 days (mean 2.8 days). Table 1 The numbers of oocysts per gram faeces (OPG) passed by four groups of three calves inoculated with 10 million, 100 million, 5 x 20 million or 400 million oocysts of Eimeria alabamensis and by three uninoculated control calves No. ofoocysts in inoculum

10 million

100 million

5 ×20 million

400 million

0

Calf OPG ( X 103) on days from first inoculation No. 1-5 6-7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

<0.5 <0.4 <0.4 <0.1 0.0 <0.9 <0.2 <1.0 <1.0 <0.9 <0.4 <0.2 0.0 <0.2 <0.4

<0.3 <4.2 <0.3 <0.2 0.0 <0.6 <1.5 <0.8 <0.3 <0.4 <1.2 <0.3 0.0 0.0 0.0

12

13

14

35000 74000 100 1.1 0.4 0.5 1.2 29000 5250 0.8 2.2 0.5 3.7 0.0 600 6700 100 8.3 0.7 0.0 0.2 600 6700 550 540 36.0 10 0.0 100 5.7 0.7 1.6 2.1 2.4 1.0 60000 29000 100 6.8 0.3 2.2 1.6 2000 9700 3800 440 120 90 8.7 400 3100 210 1250 210 120 0.0 38 1.2 50 0.8 70 50 1.5 4500 8900 3600 250 18 9.0 10 18000 16500 50 51 17 52 3.2 200 5700 200 500 2.4 10 5.5 0.0 0.0 0.0 0.0 0.1 0.3 0.0 0.0 0.7 0.4 1.7 0.5 0.9 2.3 0.2 1.9 0.6 3.3 3.3 12 6.5

15 0.0 0.0 0.8 0.0 0.2 1.7 0.4 0.0 0.1 1.3 0.5 0.8 0.0 0.8 1.2

P. Hooshmand-Rad et al. / Veterinary Parasitology 53 (1994) 23-32

27

% 30

25

20

15

10

°

contros

. . . . . . . . . Infected

J J

I

I

1

4

I

I

I

I

7

10

13

16

Days from inoculation Fig. 1. T h e m e a n ( + S D ) dry m a t t e r c o n t e n t o f the faeces o f 12 calves infected with 10 million to 400 million Eimeria alabarnensis oocysts a n d o f three u n i n f e c t e d control calves.

The dry matter content of the faeces was significantly lower during Days 4-8 than during Days 1-3 ( P = 0 . 0 0 0 2 ) . There was no significant change in the dry matter content of the faeces of the uninoculated calves. The appetite of the ten diarrhoeic calves decreased within 3-4 days after inoculation and was reduced for 3-11 days (mean 7.1 days). On Days 5 and 6, most of these calves ate almost nothing. They all appeared depressed and the diarrhoea and depression were most severe in the calves infected with 100 million or 400 million oocysts. Five days after inoculation these calves were weak and reluctant to rise, and two of those which had received 400 million oocysts showed signs of abdominal pain. Poor appetite was also noted in Calves 5 and 9, the two calves in which only a softening of the faeces was observed. The body temperature remained normal in all calves throughout the observation period. The clinical condition of the uninoculated calves was not affected. Five days before the experiment began there was no significant difference between the body weights of infected calves and the control calves (Table 2 ). From this point until 18 days after inoculation, the infected calves had a severely reduced growth rate ( P = 0 . 0 0 0 4 ) and the calves which received 5 × 2 0 million oocysts lost weight. During this period, the growth rate of the calves which were inoculated with single doses was inversely correlated to the size of the dose (Table 2 ). Subsequently, the growth rate of the infected calves recovered but at Day 71, they had still not compensated for the period of reduced growth rate. The mean ( + SD) weight gain of the control calves from Day - 5 to Day 71 was 65 ___2 kg. This was significantly ( P = 0 . 0 3 ) greater than the 56 + 4 kg weight gain made

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Table 2 Body weights and growth rates of 12 calves infected with 10 million to 400 million Eimeria alabamensis oocysts and of three uninfected controls Weight (kg)

Growth rate (g day -~ )

Day-5

Day 28

Days-5tol8

DayslS-28

Days 28-71

X

SD

X

SD

867 667 683 667 767

58 513 465 551 116

967 464 943 732 1000

78 84 229 209 112

X

SD

X

SD

X

84.3 72.3 72.3 80.0 83.7

5.7 12.5 11.0 11.5 2.5

100.7 82.7 81.5 83.7 108.0

5.8 11.6 7.5 11.9 5.6

333 160 101 -130 725

SD

Infected 10 million lOOmillion 400 million 5 × 20 million

Controls

50 351 132 157 165

Table 3 The numbers of oocysts per gram faeces (OPG) excreted by four calves challenged with 100 million Eimeria alabamensis oocysts 72 days after the first inoculation No. of

oocysts in first

Calf No.

OPG ( × 103) on days from challenge 0

1

2-6

7

8

9

10

11

12

0.1 0.0 0.2 0.3

0.9 0.6 1.7 0.9

<0.2 <0.2 < 0.1 <0.1

0 0 156 0.7

560 420 11200 4200

840 540 3000 3000

740 128 540 260

160 1.0 1.4 0.5

7.2 0.3 0.0

inoculum 10 million 400 million

1 3 11 12

by the calves dosed with 10 million oocysts, i.e. the group of inoculated calves which had the highest growth rate during the same period.

3.2. Challenge inoculation The rates of excretion of oocysts by the four calves challenged with 100 million oocysts 72 days after their first inoculation with either 10 million or 400 million oocysts are shown in Table 3. In the group initially given 10 million oocysts the peak excretion rates were lower than after the first inoculations; the peak value of OPG was less than 10% of that after the first inoculation. The four calves all had poor appetites and diarrhoea on Days 5 and 6; Calf 12 was most affected and had haemorrhagic diarrhoea and a poor appetite for longer than the other three calves. One of the calves dosed with 10 million oocysts had a severely reduced growth rate during the 15-day-period following the reinoculation and the two calves previously inoculated with 400 million oocysts lost weight during the same period (Table 4).

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Table 4 Bodyweightsand growth rates of four calveschaUengedwith 100 million Eirneria alabamensis oocysts 72 days after the first inoculation No. of oocystsin first inoculum

Calf No.

Weight on Day 71 (kg)

Growth rate after challengea (g day- l )

10million

1 3 11 12

145 136 131 107

188 938 -562 --375

400million aDays 71-88. 4. Discussion

The prepatent period and the period during which large numbers of oocysts of E. a l a b a m e n s i s were excreted during this experiment were similar to those ob-

served by other authors (Davis et al., 1955; Smith and Davis, 1965; Soekardono et al., 1975; Graubmann, 1986). The largest numbers of oocysts were excreted by the calves which received the smallest dose ( 10 million oocysts). A possible explanation for this unexpected finding is that when the numbers of sporozoites exceed a certain level they compete with each other for access to the intestinal cells of the host and cannot all complete their life cycle. This phenomenon, called the 'crowding effect' has previously been described for E i m e r i a species in chickens ( R u f f a n d Reid, 1977). Several authors have demonstrated the pathogenicity of experimental infections of E. a l a b a m e n s i s to calves. Watery diarrhoea was observed in all calves dosed with 6 million to 30 million oocysts, of which 41-81% were sporulated (Graubmann, 1986), and in calves which were excreting more than 1 million O P G after having been inoculated with 80 million to 100 million sporulated oocysts (Soekardono et al., 1975). The parasite has also been shown to cause clinical signs such as listlessness, reluctance to rise, anorexia and weight loss (Davis et al., 1955; Soekardono, 1972). However, its effects can range from a complete absence of clinical signs through mild diarrhoea (soft faeces) to death (David et al., 1955; Soekardono et al., 1975; C.C. Norton, personal communication, 1990). This wide range of effects could be due to variations in a number of factors: to differences in the susceptibility of the calves, to differences in the numbers and viability of the oocysts administered or to differences in the virulence of different strains of the parasite. Moreover, the results of Smith and Davis ( 1965 ) indicate that the method of administration of the inoculum could possibly influence its effects. The present study was designed to study the pathogenicity of a Swedish isolate o f E . a l a b a m e n s i s and thereby to investigate its possible role in the development of diarrhoea by Swedish calves when they are first turned out to pasture. The

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results were well within the range of clinical signs described above. Two of the inoculated calves had just soft faeces, whereas the other ten had watery diarrhoea. The diarrhoeic calves all had poor appetite and appeared depressed and several of them were reluctant to rise and showed signs of abdominal pain. The clinical signs were most severe in the calves which received the largest doses of oocysts, whereas Soekardono (1972) observed the opposite results. The clinical significance of the infection is further demonstrated by the severely reduced growth rates in the infected calves during the period when they showed clinical signs, a reduction for which they did not compensate during the following 2 months. During the first period after the infection the growth rate of the calves which received single doses was negatively correlated with the size of the dose (Table 2 ) as well as with the severity of the clinical signs. However, the lowest rate of growth was noted in calves which were inoculated with 5 X 20 million oocysts and in which clinical signs were not as severe as in the calves dosed with 100 million and 400 million oocysts. It has been shown that under traditional conditions of husbandry, calves may begin to excrete oocysts of E i m e r i a when they are a few weeks old and that E. alabamensis is often one of the first species to be excreted (Weinandy, 1989; Svensson, 1993). In the present study, precautions had been taken to minimise the spread of infectious diseases from other animals and it is likely that this also decreased the spread of coccidial oocysts. However, in other respects the calves were kept like conventionally reared calves. For example, the premises had not been cleaned with any disinfectant effective against Eimeria and the calves were neither gnotobiotic nor specific pathogen free. It is therefore not surprising that all the calves excreted small numbers of oocysts of Eimeria species before they were inoculated, and that some of them excreted E. alabamensis. Svensson et al. ( 1993 ) observed that many of the calves which excrete large numbers of oocysts of E. alabamensis shortly after being turned out to pasture have already been excreting small numbers while they were housed. The fact that the calves in the present study had already been introduced to the species before they were inoculated was therefore not considered to have been a disadvantage in terms of the aims of the study. However, partial immunity against the species due to previous exposure could not be excluded as a possible explanation for the low oocyst output and insignificant clinical reaction in Calves 5 and 9. A challenge inoculation with 100 million oocysts of four of the calves 72 days after their first inoculation resulted in another period of diarrhoea and the excretion of large numbers of oocysts (Table 3 ), suggesting that the first dose had not induced any substantial immunity. Davis et al. (1955 ) have previously demonstrated that a given animal may be infected several times. Two or more clear-cut infections were generally produced as a result of a series of inoculations extending over several months, and in some cases four inoculations were required before the host failed to respond; 39 clear-cut infections resulted from 58 second or later inoculations. Soekardono et al. (1975 ) observed that calves which had been infected with 10 million or 80 million sporulated oocysts of E. alabamensis ex-

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creted far fewer oocysts when they were infected with 100 million sporulated oocysts 21 days later. In the present study, the peak excretion rates of the calves previously inoculated with I 0 million oocysts were indeed lower after the challenge inoculation than after the initial inoculation, although the decrease was not as large as that reported by Soekardono et al. (1975). In the calves inoculated with 400 million oocysts, however, the reduction in excretion rate was insignificant (Table 3). This, together with the differences in growth rates between the two groups of reinoculated calves (Table 4), indicated that a dose of 10 million oocysts conferred better immunity than a dose of 400 million. The results of this study show that a large dose of the sporulated oocysts of this particular strain of E. alabamensis was pathogenic under experimental conditions. At the beginning of the grazing season, calves with diarrhoea have been observed to excrete millions of oocysts of E. alabamensis per gram of faeces. Although the output of oocysts is not a reliable indication of the intake of oocysts, this high rate of excretion strongly suggests that large infective doses of E. alabamensis are prevalent under natural conditions in Sweden. Provided that the strain used is representative of those naturally occurring in Sweden, it is therefore reasonable to consider that E. alabamensis is a potential cause of diarrhoea, reductions in growth rate and listlessness in calves at pasture. 5. Acknowledgements The study was supported financially by the Swedish Farmers' Foundation for Agricultural Research and in part by the Swedish Council for Forestry and Agricultural Research. Britt-Louise Ljungstr/Sm is acknowledged for technical assistance and Dr. C.C. Norton, Central Veterinary Laboratory, Weybridge, UK, for verifying the identity of the species of Eimeria.

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abamensis Christensen 1941 with special reference to prophylaxis in young breeding stock. ) Thesis, Humboldt University, Berlin, Germany. Ruff, M.D. and Reid, W.M., 1977. Avian coccidia. In: J.P. Kreier (Editors), Parasitic Protozoa, Vol. III. Academic Press, New York, pp. 33-69. Smith, W.N. and Davis, L.R., 1965. A comparison of dry and liquid feeds as vehicles for coccidial infection in cattle and sheep. Am. J. Vet. Res., 26: 273-279. Soekardono, S., 1972. Redescription of the life cycle of the bovine coccidium Eimeria alabamensis Christensen 1941. Thesis, Auburn University, AL. Soekardono, S., Ernst, J.V. and Benz, G.W., 1975. The prepatent and patent periods ofEirneria alabamensis and further description of the exogenous stages. Vet. Parasitol., 1:19-33. Svensson, C., 1993. Peripartal excretion ofEimeria oocysts by cows on Swedish dairy farms and the age of calves at first excretion. Acta Vet. Scand., 34:77-81. Svensson, C., Hooshmand-Rad, P., Pehrson, B., Trrnquist, M. and Uggla, A., 1993. Excretion of Eimeria oocysts in calves during their first three weeks on pasture. Acta Vet. Scand., 34:175-182. Weinandy, H., 1989. Langzeitstudie zur Epizootologie von Kokzidieninfektionen bei stallgehaltenen K~ilbern und Jungrindern. (Longterm study of the epizootiology of coccidia infections of housed calves and young cattle. ) Thesis, Justus-Liebig University, Giessen, Germany.