Enzyme variation and pathogenicity of recent field isolates of Eimeria tenella

Research in Veterinary Science /989. 46. 79-83

Enzyme variation and pathogenicity of recent field isolates of

Eimeria tenella

M. W. SHIRLEY, H. D. CHAPMAN, Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, Cambridgeshire PE17 2DA, J. KUCERA, Research Institute of Feed Supplements and Veterinary Drugs, Jilove, near Prague, 25449, Czechoslovakia, T. K. JEFFERS, Lilly Research Laboratories, PO Box 708, Greenfield, Indiana 46140, USA, P. BEDRNIK, Research Institute of Feed Supplements and Veterinary Drugs, Jilove, near Prague, 25449, Czechoslovakia

Seventy isolates of Eimeria tenella, obtained from commercial poultry farms worldwide and four reference laboratory strains were characterised by studies on the electrophoretic mobility of up to three enzymes. All populations possessed the same electrophoretic form of lactate dehydrogenase and malate dehydrogenase and one of two forms of glucose phosphate isomerase. One isolate was characterised by both forms of glucose phosphate isomerase. Studies on several isolates indicated that there was no correlation between the form of glucose phosphate isomerase found and the pathogenicity of an isolate.

Materials and methods

Experimental animals Houghton Laboratory Light Sussex chickens, which had been reared in isolators (free from specified pathogens, including coccidia), were used. For the maintenance of isolates and the production of oocysts for enzyme studies, chickens aged three to five weeks were kept in glass fibre isolators (Cooper and Timms 1972). For pathogenicity studies groups of 30, weightmatched three-week-old chickens (in three subgroups of 10) were given 2· 5 x I ()4 oocysts or kept as uninoculated controls, and weighed at four and seven days after infection. Oocysts less than two weeks old were used. The mean gains in weight were compared statistically by analysis of variance.

EIMERIA tenella is a common species of coccidium from the domstic chicken and some of the characteristics of several strains that have been maintained in the laboratory have been reported previously. Comparative investigations of strains of this species, however, are few although Joyner and Norton (1969) considered that the Weybridge strain was slightly Parasitesmore pathogenic than the Houghton strain. In The parasites studied are listed in Tables I and 2. subsequent studies on the electrophoretic mobility of The Houghton (H) and Weybridge (W) laboratory enzymes, Shirley and Rollinson (1979) and Nakamura strains of E tenella were isolated in the UK in 1949 et al (1986) found that laboratory strains of E tene/la and before 1947, respectively. The Wand Beltsville possessed one of two types of glucose phosphate (B) strains were kindly provided by Dr Joyner and Dr isomerase and a single type of lactate dehydrogenase. Doran, respectively. The Wisconsin (Wis) strain was Little is known about the characteristics of isolates isolated from the USA in about 1930. of E tene/la from the field other than, for example, Field isolates of E tenella were obtained by four of their relative resistance to certain anticoccidial drugs the authors (P.B., J.K., T.K.J. and H.D.C.) and (Chapman 1986). others were kindly provided by Dr V. Shkap, Dr. M. The work described here characterised enzymes Stallbaumer and Dr A. J. Trees. Isolates from the UK from isolates obtained from widely differing geo- were obtained from sites on which broilers, broilergraphical locations and compared them with those of breeders and replacement layers were reared. No reference laboratory strains. In addition, to attempt was made to purify the isolates by inoculation determine whether there is any correlation between of birds with a single oocyst. the enzyme type and pathogenicity, results of comparative studies on the pathogenicity of some field isolates, and two reference laboratory strains of Electrophoresis and enzyme location E tenella are given. Homogenates of oocysts were prepared and 79

80

M. W. Shirley, H. D. Chapman, J. Kucera, T. K. Jeffers, P. Bedrnik

TABLE 1: Electrophoretic variants of enzymes from strains and isolates of E tenella Parasite Laboratory Field strain isolate Beltsville (B I Houghton I HI Wisconsin (Wisl Weybridge (WI FS117 Svtzia Disson Gedera FS127 FS133 Jena Miretice Rozhovice Bylany Prisovice Velka Vysocina Xaverov FS603 FS604 FS605 FS616 FS617 FS618 FS619 FS621 FS2261 A204 FS264 FS622 FS625 FS628 FS631 FS634 FS637

USA

England USA

England Israel Israel Israel Israel Holland West Germany East Germany Czechoslovakia Czechoslovakia Czechoslovakia Czechoslovakia Czechoslovakia Czechoslovakia Czechoslovakia New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand Philippines USA USA Texas, USA Georgia, USA Arkansas, USA Delaware, USA S Carolina, USA Mississippi, USA

LDH

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

GPI

MDH

Isolate

Stock

Source in UK

PL RO

Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler Broiler breeder Broiler breeder Broiler breeder Broiler breeder Broiler breeder Broiler breeder Broiler breeder Replacement layer Replacement layer Replacement layer Replacement layer Replacement layer Replacement layer

Sussex Hampshire Hampshire Hampshire Hampshire Essex Essex Suffolk Suffolk Norfolk Norfolk Norfolk Norfolk Norfolk Cambridgeshire Cambridgeshire Cambridgeshire Cambridgeshire Cambridgeshire Warwickshire Yorkshire Yorkshire Yorkshire Yorkshire Norfolk Gwynedd Gwynedd Herefordshire Devon Devon Dyfed Oyfed Yorkshire Yorkshire

WA

PA DO

9

9 9 9 9

EL HW

BU

9 9 9 9

9

AS

BR SA US OB

1

OK

1 1

GH WH

CT

9 9 9 9

1 1 1 1 1 1 1 1 1

1+9

1

9 9

9 9 9

9 9 9

UK

Enzyme and code number

Enzyme and code number Source

TABLE 2: Electrophoretic variants of enzymes from isolates of E

tenella from the

9 9

8

OV FD RU

PU LE SH

RL FS103 FS111 FS112

HE BO WE

CO BL PI

EH AR BA

subjected to thick-layer starch gel electrophoresis as described by Shirley (1975). Zymograms were prepared for the enzymes glucose phosphate isomerase (oei) (EC 5.3.1.9), lactate dehydrogenase (LOH) (EC 1.1.1.27) and malate dehydrogenase (MOH) (EC 1.1.1.37). Two enzymes (GPI and LOH) were characterised from all isolates/strains, but not all isolates were studied for MOH. Numbers were arbitrarily assigned to the enzyme variants as described by Shirley and Rollinson (1979). Results

Enzyme electrophoresis Results are summarised in Tables I and 2 and Figs I and 2. A single form of LOH (coded LOH-8) and MOH (coded MOH-!) was found in all the isolates examined (Figs Ib, c and 2c, d). In contrast, two forms of GPI were found (coded GPI-! and GPI-9). The B, Hand Wis strains and six isolates from the USA were charac-

CH DA YO

TO

LDH

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

8

8 8 8 8 8 8 8 8 8 8

GPI

9 9 9

9 9 9 9 9 9 9 9 9

9 9 9 9

9 9 9 9 9 9 9 9 9 9 9 9 9 9

9 9 9 9

Sussex

8

9

Suffolk

8

9

Suffolk

8

9

Yorkshire

8

9

Yorkshire,

8

9

Fife

8

9

terised by variant GPI-! which migrated the shortest distance in the gel. The W strain and 62 of the 69 field isolates possessed a form of the enzyme (GPI-9) which migrated further from the origin. Only one isolate (FS621) was characterised by both variants of OPI. Variants of GPI in some of these strains and isolates are shown in Figs la and 2a, b.

Pathogenicity of recent isolates There were no significant differences in the pathogenicity to chickens (as determined by bodyweight gain) of isolates characterised by GPI-! or GPI-9

Enzyme variation in E ten ella a

~6a·······'

--- --1

2

b

••

'-...-' 1

c

'-...-' 1

'-...-' 3

'-...-' 4

'-...-'

'-...-'

'-...-'

2

1

4

'-...-' 3

'-...-' 1

.......... '-...-' 5

'-...-' 6

--- --- --- --2

7

8

TABLE 3: Me!'n bodyweight gain and mortality of Light Sussex chickens after inoculation with oocysts of the H or W strains or field isolates of E tenella GPI

Experiment number

9

FIG 1: Zymograms of: (a) glucose phosphate isomerase, (b) lactate dehydrogenase and (c) malate dehydrogenase. 1 Miretice; 2 Houghton; 3 Jena; 4 Rozhovice; 5 Vysocina; 6 Xaverov; 7 Bylany; 8 Prisovice; 9 Velka

(Table 3). There were differences between isolates in lethal effects but these were not correlated with enzyme type.

Parasites given*

reference number

Mean bodyweight gain (g)t

Percentage mortality

5*

H W Bylany Miretice Priscovice Velka None

1 9 9 9 9 9

6·2a 6·3a 10·40 -6'3a 0'2a 2·5a 51·4b

25 7 43 27 27 0

2

H W FS628 FS631 FS634 F637 None

1 9 1 1 1 1

-7'la 0·8a -10'8a -3'8a -10'9a -16·8a 41·9b

25 35 46 43 36 36 0

3

H W FS616 FS617 FS622 FS625 None

1 9 9 9 1 1

-0'5a -9'la -14'2a -12'Oa -10'6a -1'6a 45·3b

20 45 53 37 43 13 0

'-...-' 1

.... --.

81

• 2·5 x 1()4 sporulated oocysts inoculated orally t Measured between days 4 and 7 after inoculation of oocvsts, Standard error of treatment means = 3·74 '" 20 birds only in this group; 30 in all the others a.b Values not followed by a common letter are significantly different (P<0·051

electrophoretic vananon of coccidial enzymes and extend those recently described by Shirley (1986). All the strains and isolates of E tenella were characterised by the same variants of LDH and MDH, whereas intraspecific variation was found with GPI. However, it was necessary to bite cautious in the interpretation of gels stained for GPI because subbands which migrated towards the anode from the main band were occasionally seen (Figs la and 2a). This problem was also recognised by Carter (1978) who recommended that the main band nearest the cathode be used to indicate the location of this enzyme. To minimise the presence of subbands of GPI which occur readily in stored material, only freshly prepared homogenates from fresh cultures of oocysts were used in this study. The B, Hand Wis reference laboratory strains and six of the isolates from the USA possessed GPI-1. The most common form of the enzyme was GPI-9 which was found in the W strain and most of the field isolates, including all of those from the UK, Europe and New Zealand. Surprisingly, only one isolate (FS62! from the Philippines) possessed both GPI-I and GPI-9.

Discussion The results provide further information on the

The reason for the comparative rarity of isolates with both GPI-l and GPI-9 is not known. It is of interest to note that the results of similar, but less extensive,

M. W. Shirley, H. D. Chapman, J. Kucera, T. K. Jeffers, P. Bedrnik

82 a

.6.,.••••••••,.•.

--- --- --- --- --2

1

b

3

4

5

2

6

4

3

5

. . - -. -. •

•••••• -• • •

....,.. •••• 5

c

8

4

9

2

10

3

14

11

13

12

15

16

4

17

8

5

5

4

9

2

10

3

11

12

13

14

-• 14

15

16

17

d

••••••••••••••• 4

2

3

14

15

16

17

4

2

3

14

15

16

17

4

2

FIG 2: Zymograms of: (a and b) glucose phosphate isomerase, (c) lactate dehydrogenase and Id) malate dehydrogenase. 1 = FS616; 2 = FS622; 3 = FS625; 4 = Houghton; 5 = Weybridge;

6= FS617; 8= FS621; 9=BA; 10=OB; 11=PU; 12=TO; 13=WA; 14= FS628; 15=FS631; 16 = FS634; 17 = FS637

Enzyme variation in E tenella studies on E acervulina and E mitis (species also characterised by intraspecific variation for GPI) showed that isolates with a combination of different types of GPI are relatively common (Shirley 1986). The greater preponderance of isolates characterised by GPI-9 is also not understood. One explanation could be that parasites possessing this variant have a selective advantage over those characterised by GPI-I. In a study on four of the field isolates (GH. AS, FD and HW) characterised by GPI-9, Chapman (1986) found that they were more pathogenic than E tenella H (GPI-I). Joyner and Norton (1969) also considered that the W strain (GPI-9) was more pathogenic than the H strain. Contrarily, in this study there was no significant difference between the pathogenicity of the Hand W reference laboratory strains, or between field isolates characterised by GPI-I or GPI-9 (Table 3). A second possibility is that a favourable mutation may have occurred at a genetic locus close to that of GPI-9 and that the selection of this trait resulted in the coselection of the enzyme variant. Populations characterised by GPI-9 or GPI-I may differ in their virulence after serial passage in embryonated eggs. The Houghton strain (GPI-I) was substantially attenuated after 20 passages (Long 1972) whereas, in similar studies (Gore et a11983, Bedrnik et al 1986), two strains which possessed GPI-9 (Shirley 1986) remained highly virulent. Acknowledgements We wish to acknowledge the excellent assistance of

83

Mrs M. A. Bellatti, Mrs M. Shaw and Mr P. Townsend. References BEDRNIK, P., KUCERA, J., JURKOVIC, P. & FIRMANovA (1986) Research in Avian Coccidiosis. Proceedings of the Georgia Coccidiosis Conference. Eds L. R. McDougald, L. P. Joyner and P. L. Long. University of Georgia Press. pp 510-525 CARTER, R. (1978) Parasitology 76, 241-267 CHAPMAN, H. D. (1986) Research in Veterinary Science 41, 281-282 COOPER, D. M. & TIMMS, J. R. (1972) Avian Pathology I, 45--57 GORE, T. c., LONG, P. L., KOGUT, M. & JOHNSON, J. (1983) Avian Diseases 27,569-576 JOYNER, L. P. & NORTON, C. C. (1969) Parasitology 59, 907-913 LONG, P. L. (1972) Journal ofComparative Pathology 82, 419-437 NAKAMURA, T., KONISHI, T. & KAWAGUCHI, H. (1986) Japanese Journal of Veterinary Science 48, 587-590 SHIRLEY, M. W. (1975) Parasitology 71, 369-375 SHIRLEY, M. W. (1986) Research in Avian Coccidiosis. Proceedings of the Georgia Coccidiosis Conference. Eds L. R. McDougald, L. P. Joyner and P. L. Long. Univesity of Georgia Press. pp 13-35 SHIRLEY M. W. & ROLLINSON, D. (1979) Proceedings of the 17th Symposium of the British Society for Parasitology. Eds A. E. R. Taylor and R. Muller. Oxford, Blackwell Science Publications. pp 7-30

Received February 2, /988 Accepted March 3, /988