Detection and assessment of sublethal infections of Eimeria tenella and Eimeria necatrix

EXPERIMENTAL

PARASITOLOGY

Detection

and

9,

243-249

(1960)

Assessment of Sublethal tenella and Eimeria L. P. Joyner

Ministry

of Agriculture,

(Submitted

and

Fisheries and Weybridge,

Infections necatrix

of Eimeria

S. F. M. Davies Food, Central England

for publication,

Veterinary

29 November

Laboratory,

1959)

1. The estimation of the packed red-cell volume by the micro-hematocrit technique proved to be a convenient method of measuring anemia in chickens infected with E. tenella or E. necatrix. 2. Hematocrit measurements were more sensitive to the effects of infection with E. tenella than observations on the growth rate. 3. With E. necatrix, a marked retardation of growth occurred in birds in which only a mild anemia was detectable.

Most of the quantitative laboratory studies on coccidiosis in chickens have been based upon mortality in controlled groups of chicks following infection with relatively large numbers of oocysts. Such experiments have permitted valid conclusions to be drawn on the production of immunity and on the therapeutic properties of drugs but they are open to criticism on the grounds that under field conditions, infection does not always terminate in death. Sublethal infections, however, frequently cause economic loss and experimental studies of them are often desirable. The observations recorded in this paper were an attempt to establish methods of detecting sublethal infections with sufficient precision to provide a basis for future experiments. The initial experiments were carried out with Eimeria tenella, the cause of cecal coccidiosis. The different stages in its development can be accurately forecast, and Natt (1959) has listed the physiological changes in the host which accompany its development. It has been shown that although it is not the only change which occurs, the loss of blood which accompanies the development of the second-generation schizonts of Eimeria tenella is the main cause of death (Natt and Herrick, 1955, 1956; Waxlcr, 1941). It was decided there243

fore to examine a number of hematological methods to investigate the conditions under which sublethal infections could regularly be detected by the measurement of anaemia. The observations were extended later to Eimeria necatrix, a species which produces in another part of the intestine, lesions which also are characterized by hemorrhage. Coccidiosis is predominantly a diseaseof the young stock and it occurs when the growth rate is of particular importance. It is mainly for this reason that growth of the infected birds is sometimes taken into account in assessingthe efficacy of coccidiostatic drugs. With both species, therefore, an attempt was made to relate hematological effects to retardation of growth. METHODS

The Rhode Island Red or White Leghorn x R.I.R. chicks used in the experiments were reared in heated wire floored hovers and fed a standard chick ration, the composition of which is indicated in Table I. They were received when one day old and kept in isolation until required. The chicks were infected with either Eimeria tenella or Eimeria necatrix by the oral administration of measured volumes of standardized suspensions of oocysts. Because relatively small numbers of oocysts were given, reproducible results depended

JOYNER AND DAVIES

214

TABLE The

Composition

I of Chick

Maize. Barley........................ Wheat feed................... Fish meal. Meat meal. Decort. groundnut meal. Soya bean meal. Lime. Calcium phosphate. . Salt. _. Vitamin-mineral concentrate.. (“Supercon Special,” Vitamins

Mash

25% 2235% 30% 5% 5%

am 7X% 20 lbs. 20 lbs. 11 lbs. 5 lbs. Ltd.)

per per per per

ton ton ton ton

upon the preparation and standardization of the cultures. In the early stages of the investigation, existing methods were modified in an attempt to improve their accuracy. Records of experiments with several species of Eimeria suggestedthat oocysts which had been separated by saturated sodium chloride floatation suffered some degree of deterioration. Disintegration of the oocysts and irregular sporulation were reduced when the use of salt was avoided. Oocysts of E. tenella were extracted from cecal tissue by emulsifying in 2% potassium bichromate in a mechanical blender, 20 ml of the solution being added to each gram of tissue up to a maximum of 2,500 ml. The emulsion was passedthrough muslin and 2% sodium bicarbonate added in order to reduce the deposition of fat and mucus. After allowing to sediment overnight, the supernatant was siphoned off and the sediment passed in sequence through three seives of 100, 200 and 300 meshesper linear inch, respectively. The seives were mounted one above the other in a specially made funnel fitted to a Buchner flask. Gentle suction was applied and the filtrate and washings were collected in the flask. The oocysts in the filtrate were allowed to sediment in oneliter cylinders, and resuspended in 2% potassium bichromate to give a final concentration of approximately lo6 oocysts/ml. Oocysts of E. necatria were extracted from

cecal

tissue

in a mechanical

blender,

but after passing the emulsion through muslin the filtrate was allowed to pass directly without suction through a single 300 mesh

sieve. The final concentration of oocysts in 2% potassium bichromate was approximately 300,00O/ml. The cultures so obtained were distributed in shallow layers in large dishes and allowed to sporulate at room temperatures. The suspensions of oocysts were standardized by estimating the numbers of oocysts present and the proportion of them in which sporulation was completed. One in 10 dilutions were counted in a Fuchs-Rosenthal hemacytometer and an average of 10 counts was taken. Sporulation was assessedby examining approximately 300 oocysts in coverslip preparations under a l/6-inch objective. The oocysts were stored at +5”C and renewed at intervals of not more than one month. HEMATOLOGICAL

METHODS

Red blood cells were counted in a Thoma hemacytometer chamber after diluting l/200 in 1% formalin in 3% sodium citrate. Hemoglobin was estimated by the alkaline hematin method of Clegg and King (1942). The color densities were measured photoelectrically and converted to grams of hemoglobin/100 ml by means of a calibration curve obtained with standard hemin solutions. The method appeared to be satisfactory, although little appears to be known about its accuracy for avian blood. In the present comparative experiments, it proved to be quick, centrifugation or filtration being unnecessary, and it gave values comparable with published data for normal chicks. Packed cell volumes were originally determined by the Wintrobc hematocrit method using oxalated blood. This was later modified by using heparin as the anticoagulant. The method however, suffered from the disadvantage that relatively large volumes of blood were necessary for each determination

and repeated

determinations

on

young chicks were not possible. The microhematocrit method using capillary tubes and a high speed centrifuge (Hawksley) overcame

this

difficulty

and

enabled

daily

duplicate readings to be obtained with approximately 0.1 ml of blood after ccntrifugation

for 10 minutes.

SUBLETHAL

245

COCCIDIOSIS

In the group which received 50,000 oocysts two birds died on the 5th day and a The Measurement of Anemia following third died on the 6th day after infection. In Infection with E. tenella the group which received 10,000 oocysts, one Three methods of recording blood changes death occurred on the 6th day after infeclikely to arise from severe hemorrhage were tion. With both these groups therefore the available and the experiment recorded in subsequent readings were recorded as averFig. 1 was designed to compare the results ages for the survivors. It will be seen from Fig. 1 that the hemowhich they gave when chicks were infected with different numbers of oocysts. globin concentration and the packed cell Twenty-five chicks aged 35 days were di- volume were the most sensitive. With both vided into five groups and their blood was these methods a marked fall was recorded examined on nine occasions following infor all four levels of infection. The red-cell fection with 1,000, 5,000, 10,000 or 50,000 count, however, revealed a marked fall only oocysts respectively. The fifth group was after infection with 50,000 oocysts, a level uninfected and served as normal controls. of infection sufficiently heavy to cause apRESULTS

&E

IO -

8 0 a. 6 f

864-

0

2

Time

FIG. 1. Changes

in the blood

8

6

4

in

of chicks

lo

Days infected

with

E. tenella.

12

246

JOYNER

AND

preciable mortality. The hematocrit method was chosen for subsequent experiments. Only small volumes of blood were necessary for the micromethod and its rapidity enabled a series of birds to be examined in a short period of time. Anemia and Depression of Growth Rate following Infection with E. tenella From the previous experiment it was evident that packed red-cell volume and hemo-

DAVIES

globin determinations were the most sensitive to the changes accompanying E. tenella infections. It remained to establish whether infections which were severe enough to cause measurable anemia also reduced the growth rate of the birds. The experiment recorded in Fig. 2 was carried out with 6-week old birds divided into four groups of nine. One group was uninfected and the other three received infec-.&+-

+ -0

CONTROLS 2000 OOCYSTS 5000 OOCYS-iS 10000 OOCYSTS

I

II O-l

0

I

2

3

4

NUMBER

O-I

0

I

2

3 MJMBER

FIG.

2.

The

packed

red cell volume

5

6

7

OF DAYS AFTER

4

s

6

OF DAYS AFTER

and the growth

8

9

IO

II

12

lo

II

12

lN=ECTlCN

7

8

9

WECTION

of chicks

infected

with

E. tenda.

SUBLETHAL

217

COCCIDIOSIS

tions of 2,000, 5,000 or 10,000 oocysts, respectively, per bird. The growth of the uninfected control birds showed a steady increase during the 12 days of the experiment. The birds receiving 2,000 or 5,000 oocysts showed slight retardations of growth at about the 5th or 6th day after infection, but the only group to show a clearly defined fall in the growth rate was that receiving 10,000 oocysts. In this group, however, one bird died on the 5th day and three died on the 6th day after infection. In the surviving birds of this group growth was resumed after the 7th day.

The hematocrit curves showed a distinct drop between the 4th and 7th days in all three infected groups. By the 12th day, normal values were almost regained.

Anemia and Depression of Growth Rate following Infection with E. necatrix In the experiment recorded in Fig. 3, three groups of six 5-week old birds were infected with 1,000, 10,000 and 20,000 sporulated oocysts of E. necatriz respectively per bird. A similar group of six birds remained uninfected. Hematocrit readings and weight gains during t,he course of the experiment were recorded. --t-CONTROLS * 1000 -c- 10000 t 20000

NUMBER

L o-2

0

I

2

3 MMBER

FIG. 3.

The

packed

red cell volume

Cf DAYS AFTER

INFECTION

4

7

5

6

OF DAY5 AFTER

and the growth

ll

9

IO

OOCYSTS OOCYSTS oOCYSTS

II

I2

INFECTION

of chicks

infected

with

E. necc&z.

248

JOYNER

A gradual drop in the packed cell volume was noted in all groups but unlike the birds infected with E. tenella (Fig. 2) there was no distinct drop between the 4th and 7th days. Birds infected with 1,000 oocysts and those which remained uninfected showed a steady gain in weight during the experiment. Those which received 10,000 or 20,000 oocysts showed a marked reduction in weight during the 5th to the 10th days after infection. DISCUSSION

The only available method of varying the severity of a coccidial infection in a controlled experiment is to administer different numbers of parasites, but it is difficult to standardize precisely a given suspension of oocysts. An estimation of sporulation involves subjective judgment and the error in counting small numbers of oocysts is likely to be appreciable. The method of storage and the age of the oocysts are other variable factors. Although an effort has been made to define the methods used in the present study as precisely as possible, conditions in other laboratories may produce slightly different results. The infections used in these experiments were induced in chicks aged 4 to 6 weeks using estimated doses of 1,000 to 20,000 oocysts. With this level of infection occasional deaths may occur but with levels only slightly above this, appreciable mortality may be expected. With both E. tenella and E. necatrix, therefore, the present observations are related to roughly comparable infections, i.e., those which just fail to kill. Cecal coccidiosis is usually a severe condition with which a wide variety of changes are associated. Because hemorrhage in the ceca forms a major part of the disease, it would be expected that the most marked changes would occur in the blood. Other changes however have been described which arise from different effects upon the host’s metabolism. These include changes in the blood sugar (Pratt, 1940), a decrease in muscle glycogen (Pratt, 1941), interference with total metabolism and temperature regulation (Herrick, 1950) and a reduction in t,he ability of the muscles of the bird to do

AND

DAVIES

mechanical work (Levine and Herrick, 1954). The present experiments confirm, however, that with E. tenella the effects arising from hemorrhage form a sensitive indication of the course of the infection. Not only could the anemia be demonstrated when death did not occur, but as shown in Fig. 1, a reduction in the packed red cell volume could be detected even when effects upon general metabolism were not sufficiently severe to retard growth. With E. necatrix, however, measurement of growth provides a more sensitive standard. The experiment recorded in Fig. 3 showed a marked retardation of growth in groups of birds in which only mild anemia was detectable. This is in agreement with observed differences in the pathology of the two infections. Although the main pathological effects of infection with both species may be associated with the development of the late schizonts, it is only in the more severe infections with E. necatrix that hemorrhage is marked; whereas with E. tenella, loss of blood from the ceca appears to occur when the infection would be otherwise undetectable. Furthermore, damage to the intestinal mucosa, the site of the schizonts of E. necatrix, is more likely to affect the general metabolism of the host than is damage to the ceca, to which E. tenella is confined. It must be remembered in designing experiments based upon these standards, that they are dependent upon different conditions. For example, observations on E. necatrix based on growth, would be dependent upon a balance between impaired intestinal function, which may extend over a considerable period, and the host’s ability to make compensations in its metabolism. Such a balance could easily be affected by factors not directly related to the activity of the parasite such as food palatability, or a sudden fall in room temperature. For E. tenella however, observations on anemia depend upon the hemorrhage caused by the parasite between 48 and 120 hours after infection, and the host’s ability to replace the lost blood. Such a balance would not be so dependent upon environmental factors although in chemotherapeutic experiments, for example, drugs with anti-

SUBLETHAL

hemopoietic effects might cific changes.

induce non-spe-

&KNOWLEDGMENT is due to Dr. S. B. Kendall for his assistance in the initial design of this research. Acknowledgment

REFERENCES J. R., AKD KING, E. J. 1942. Estimation of haemoglobin by the alkaline haematin method. Brit. Med. J. II, 329. HERRICK, C. A. 1950. The effect of coccidia on the metabolism and temperature regulation in chickens. Poultry Sci. 29, 763. LEVISE, L., .4SD HERRICK, C. il. 1954. The effects of the protozoan parasite Eimeria tenella on the ability of the chicken to do muscular work when its muscles are stimulated directly and indirectly. J. Parasitol. 40, 525-531. NATT, M. P. 1959. The effect of cecal coccidiosis on the blood cells of the domestic fowl. 3. The changes in the leucocyte picture during the course of the infection. Exptl. Parasitol. 8, 182-187. CLEGG,

COCCIDIOSIS

249

M. P., AND HERRICK, C. -4. 1955. The CffeCt of cecal coccidiosis on the blood cells of the domestic fowl. I. A comparison of the changes in the erythrocyte count resulting from hemorrhage in infected and mechanically bled birds. The use of the hematocrit value as an index of the severity of the hemorrhage resulting from the infection. Porcltry Sci. 34, 1100-1106. NATT, M. P., AND HERRICK, C. A. 1956. The effect of caecal coccidiosis on the blood cells of the domest,ic fowl. 2. The changes in the blood volume during the course of the infection. Poultry Sci. 35, 311-316. PRATT, I. 1940. The effect of E. tenella upon the blood sugar of the chicken. Trans. Am. Microstop. sm. 59,31-37. PRATT, I. 1941. The effect of E. fenella upon the glycogen stores of the chicken. Am. J. Hyg. 34c, 54-61. WAXLER, S. H. 1941. Changes occurring in the blood and tissue of chickens during coccidiosis and artificial hemorrhage. Am. J. Physiol. 134, 19-26. WATT,