Organ and tissue distribution of the exoerythrocytic stages of various avian malarial parasites

EXPERIMENTAL

Organ cytic

PARASITOLOGY

6, 143-162 (1957)

and Tissue Distribution Stages of Various Avian

of the Malarial

ExoerythroParasites

Clay G. Huff* Naval

Medical (Submitted

Research Institute, for publication,

Bethesda, 25 July

Maryland 1956)

The discovery of exoerythrocytic stages of malarial parasites has raised many new questions in regard to the interrelations het,ween t,ho host cells and the parasites. Foremost among these is the manner 1)~ which changes are wrought in t,he relationship between the parasites and the cells of the host. Because of changes in the host, in the parasite, 01 in both, host cells which at one stage in the infection are not, parasitized are invaded at a later stage of infection. In this paper I shall present the results of histologic examination of sections of birds killed at close intervals of time following inoculations of sporozoites and of erythrocytic stages of several species of avian malarial parasites. These results are presented with the view toward delineating more definitely the problem mentioned above and with the hope that they will offer some clues toward its solution. MATERIALS

AND

METHODS

Parasites The following species of avian Plasmodium were used: cathemerium, circumjlexum, elongatum, fallax, gallinaceu,m, lophurae, and relicturn. In all except relicturn, only one strain of each was used. The Coatney, or 1P, strain of P. relicturn adapted to pigeons and the 1Pl-1 strain adapted to canaries were used. * The author wishes to thank the following persons for their invaluable assistance: Mrs. Dorothy Marchbank, Mrs. Henrietta Lucke, Miss Tsugiye Shiroishi and Mr. D. G. Hartlieb, HMl, USN. The opinions or assertions contained herein are the private ones of the writer and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 143

144

CLAY G. HUFF

Host Animals The following birds were utilized: canaries, chickens, chick embryos, ducks, gosling embryos, partridges, pheasants, pigeons, quail, and turkeys. The partridges were Alectoris graeca chukar; the pheasants, Phasianus colchicus; and the quail, Colinus virginianus. Methods No special technics were used beyond those described by Huff and Coulston (1944). Sporozoites of P. gallinaceum were produced in Aedes aegypti; of P. fallax in A. albopictus; and of P. cathemerium and P. relicturn in Culex pipiens. In much of the earlier work more extensive searching for exoerythrocytic stages in all of the organs of the host was carried out. Experience showed that the organs which contained them most frequently were: spleen, liver, heart, brain, and lung. Consequently searching for the exoerythrocytic stages was usually limited to these organs plus kidney, which served as an indication of whether or not the invasion was general over all organs and tissues. Extensive studies had previously been done 011P. elongatum by Huff and Bloom (1935). Further studies were included here only for comparison with the other species of parasites. The inoculums used in sporozoite-induced infections consisted of the macerated salivary glands from 25-50 infected mosquitoes. No sporozoite-induced infections of P. lophurae, P. elongatum, or P. circumflexurn were included because no strain of lophurae capable of infecting mosquitoes was available and in the case of the other two species, the mosquitoes available were not good vectors. RESULTS

Relation of Inoculum Size to Formation of E’xoerythrocytic Stages One of the variables most easily controlled in the host-parasite relationships was the size of inoculum administered. The validity of many of the data to be presented in this paper would be lessened if it were shown that the numbers of parasites inoculated influenced the number, distribution, and time of appearance of the exoerythrocytic stages. The following experiments were carried out to answer this question. Two series of experiments were carried out on blood-induced infections, the first employing P. gallinaceum in chickens and the second P. jallax in turkeys. Birds were inoculated in triplets or pairs, each bird of a group receiving inoculums of different size. In each case the birds were killed from 3 to 15 days after inoculation, and the principal organs sectioned

EXOERYTHROCYTIC

STAGES

OF AVIAN

TABLE

145

PLASMODIA

I

Relationship Between Size of Inoculum and Presence and Distribution of Exoerythrocytic Stages in Blood-Induced Injections Exoerythrocytic

Age of infection (days)

Brain

Size of inoculum

P. gallinaceum

3-5 7 9 11 13 15

3 3 3 8 8 8

x x x x x x

106-2 x 106-3 x 106-3 x 106-2 x 106-S x 106-8 x

108 107 106 108 107

-

107

P. jallax

3 5 7 9 11-13 15

lo”107 2 x 106-2 x 107 2 x 106-2 x 106 lo”108 2 x 106-2 x 10” 106

+

Heart

Kidney

stages in Liver

Lung

Sphl-

in chickens + +a + +

+

+

-

+ +” +” +

+

-

+

+

-

-

-

f"

in turkeys +

a Abundant at 3 X 108. b Abundant at 2 X 10’. c sparse at 8 x 10’.

and stained. Table I presents the results from the two experiments. No differences in the time of appearance or the distribution of exoerythrocytic stages were reflected in the birds receiving inoculums varying as much as one hundredfold. Counts of exoerythrocytic stages per unit of volume of tissue were not made. However, a few instances of noticeably greater or lesser abundance were recorded in some of the organs of chickens infected with P. gallinaceum. These show some indication that the abundance of exoerythrocytic stages might have been greater in the organs of birds receiving the highest inoculums. However, this point needs to be given quantitative study. Distribution

of Exoerythrocytic Stages of Various Species of Avian Malaria in Different Hosts

Success has been attained by various authors in establishing parasitemia by several avian malarial parasites in hosts that are rather distantly separated systematically. In fact McGhee (1951) was successful

146

CLAY

G. HUFF

in maintaining the avian parasite, P. lophurae, in baby mice for many passages. Moreover Huff (1948), working with P. relicturn in pigeons and doves found in general that more species of hosts could be infected with inoculations of infected erythrocytes than of sporozoites. For this reason no attempt has been made to find exoerythrocytic stages of any of the parasites used in hosts in which parasitemia could not be established by the inoculation of infected erythrocytes. In the following results it can be assumed, unless otherwise stated, that parasitemia was established in the animals the tissues of which were examined for exoerythrocytic stages. Plasmodium gallinaceum in chickens. The distribution of exoerythrocytic stages in chicks following inoculations of sporozoite suspensions as well as the corresponding information on blood-induced infections is given in Table II. More intensive studies of their distribution in the earlier portions of sporozoite-induced infections were made by Huff and Coulston (1944) and Coulston, Cantrell, and Huff (1945). Depending somewhat upon the size of the inoculum the exoerythrocytic stages were to be found 2 or 3 days after the inoculation of sporozoites. Huff and Coulston (1944) showed that mature exoerythrocytic stages could be demonstrated in the spleen 36 hours after massive inoculations (sporozoites from 200 mosquitoes inoculated into a small chick). No exoeryth-

Distribution

TABLE II of Exoerythrocytic Stages of Blood-Induced and Sporozoite-Induced Infections of Plasmodium gallinaceum in Chicks

Age of infections in days (number of birds used)

Brain

Heart

Kidney

Liver

Lung

Spleen

Intestine

Tbymus

Sporoeoite-induced 2 (5) 3-10 (27) 49-52 (2)

+;12)

+&

+--02)

+;121

+

(12) +’ z,

-

Blood-induced

13~9 (6) 3-6 8-9 10-11 13-15 17-18

(53) (4) (6) (3) (2)

-

-

-

-

-

-

+-& + (3) + (4) + (1) + (1) + (2) + (1) -

+(l) -

-

+ (4) + (1)

+ + + +

(12) (1) (6)

(2) + (2) + (2)

+

(4) + (2)

EXOERYTHROCYTIC

STAGES

OF AVIAN

PLASMODIA

147

rocytic stages could be found in other organs prior to 48 hours following inoculation of large doses of sporozoites (Huff, 1951). The results shown in Table II confirm the previous results. Three days after intravenous inoculations of sporozoites exoerythrocytic stages were found in the vascular endothelium of most of the organs. The fact that they were not found in a particular organ of all birds examined after a given interval is probably to be explained upon inadequate sampling. The occurrence of exoerythrocytic stages in birds inoculated with infected erythrocytes was markedly different from what it was in sporozoite-induced infections. With the exception of the finding of these stages in the liver of one bird 4 days after inoculation their distribution did not become general until the tenth post-inoculation day. The distribution of the exoerythrocytic stages in the two series of infections (erythrocytic and exoerythrocytic) differed essentially in the delayed occurrence of the stage at which most of the organs were infected. This delay was exactly 7 days (Table II). Plasmodium gallinaceum in turkeys. The turkey was not as susceptible to erythrocytic infection with this species of parasite as the chicken. Following the intravenous inoculation of the sporozoites from 30 infected mosquitoes only a light parasitemia resulted. However, the parasitemia following the inoculation of infected blood reached peaks ranging between 15 and 70% of the red cells. This relationship is also reflected in the less abundant exoerythrocytic stages when compared to the numbers found in the chick. Moreover the exoerythrocytic stages were longer appearing and at no time did they become as widely distributed in the turkey (Table III) as in the chick. Contrary to what the situation was in the chick there was no clear preference for the spleen early in the infection. The distribution of the exoerythrocytic stages in turkeys which received inoculations of erythrocytic parasites followed rather closely the pattern found in the chick. For the first 11 days no organ except spleen was found to be infected and at 16 days some of the other organs were beginning to harbor the tisue stages (Table III). P. gallinaceum in other hosts. Ducklings were highly insusceptible to infection, as measured by parasitemia, with P. gallinaceum whether the inoculations were made with sporozoites or with erythrocytic stages. Exoerythrocytic stages were sought in the tissues only from animals inoculated with sporozoites. They were found only in the spleens of two out of ten animals (see Table IV) 5 days after inoculation.

148

CLAY

G.

HUFF

TABLE III Distribution

of Exoerythrocytic Stages in Sporozoite-Induced and Blood-Induced Injections of Plasmodium gallinaceum in Turkeys

Age of infection in days (number of birds used)

Brain

Heart

Kidney

Liver

Lung

Spleen

Sporozoite-induced 2-4 5-6 6-10 12-14

(6) (4) (6) (4)

16 (2)

-

-

-

-

-

+(2) + (3) + (3) + (2)

-

-

-

-

+ + +

(1) (2)

(2)

+

(1)

+

-

(1)

+ + + +

(3) (4) (1) (1)

Blood-induced 4 (1) 5-11 (5) 14 (1)

16 (2)

+

+

(1)

+

(1)

-

-

-

+

-

-

-

+

(5) + (1)

Parasitemia resulted in chick embryos inoculated either with sporozoites or erythrocytic stages. As shown in Table IV, exoerythrocytic stages were found in heart, spleen, and liver of 3 birds out of 10 of bloodinduced infections and in the spleen only of 1 out of 8 sporozoite-induced infections. Light to moderate parasitemia resulted from blood-induced infections of pheasants. Exoerthrocytic stages were found in the hearts and lungs of 2 out of 17 infections (Table IV). No exoerythrocytic stages were found in any organ of 6 partridges which were inoculated with sporozoites. Two of each were killed on the 4th, 5th, and 6th days after inoculation. About half the birds inoculated with sporozoites exhibited no parasitemia and in the others the parasitemia was very slight. Plasmodium jallax in turkeys. The susceptibility of turkeys to P. jallax equalled or surpassed that of chickens to P. gallinaceum. The results from sporozoite-induced infections of the former were very similar to those of the latter (Table V) in (1) the early appearance of exoerythrocytic stages in the principal organs and (2) their disappearance after about ten days. The results from blood-induced infections of P. jallax in turkeys (Table V) are also very similar to those in similarly induced P. gallinaceum infections in chickens (Table II) in the late appearance of exoerythrocytic

Combinations

of parasite

Abbreviations: S = sporozoites; * Evidence of degeneration.

relicturn (1P) cathemerium circumJlexum

fallax fallax fallax fallax fallax fallax lophurae relictum relicturn

Br

B B S S B S B S B B B B

Chick Canary Canary Quail Pheasant Pheasant Chick Canary Canary Pigeon Canary Canary

P. P. P. P. P. P. P. P. P. P. P. P.

B = blood;

S

gallinaceum fallax fallax fallax fallax

gallinaceum

gallinaceum

Chick

P. P. P. P. P. P. P. P. P.

P. fallax

= brain;

2 0 1 1 0 0 0 1 1 2 1 0

2

2 2 3 1 0 0 1 0 0

IV

= liver;

Lu

= lung:

Lv, Lu, sp SP Br, H, Sp Lv, Lu, sp

3 11 12, 15 5 K = kidney;

H SP

8 6

Sp*

SP

Lv

Organs

present

SP H, Lu Sp, H, Lv SP

stages

1 16* 6, 18*

5

12

5 8 4, 5, 7 6

Age of infection

Exoerythrocytic

Sp

=

after

inoculation

absent

% E z

3, 7, 9, 11, 12, 15, 17 4, 10 4, 8, 10, 12, 14, 16 4, 6, 8 6, 8, 10 3-5, 8, 10, 12, 14, 16 135, 2, 4-6 4-8, 10, 16 4, 6, 8, 10, 14-16, 18 4-10, 12, 14, 16, 18 2, 4, 6, 8, 10, 12, 14

z W

E F

2 $

8

3 =; 8

3 3 B 8 3 ki

m

2, 4-16, 18, 19

3-15, 17, 21

3-6, 8, 10, 12, 14, 17 2, 4-6, 8 2, 3, 6 4-6 3-9, ll-15,17-20 4-6, 8, 10, 14, 16, 18, 19 6, 8, 10, 13, 14, 16, 20 6, 8, 10, 13, 14, 16, 16

Spleen.

22 9 2 7 3 3 9 5 13 8 13 16

15

8 15 7 7 6 18 11 9 7

Days

stages

in the Organs of the Hoeis Exoerythrocytic

No. of .Ulilll&

Stages Were Very Rare or Not Pound

TABLE

No. of animals

H = heart;

Inoculum

S B B S S S B S B

Host

in Which Exoerythrocytic

Duckling Pheasant Chick embryo Chick embryo Partridge Duckling Duckling Pigeon Pigeon

gallinaceum gallinaceum

Species

Host-Parasite

150

CLAY

G. HUFF

TABLE Distribution

V

of Ezoerythrocytic Stages in Sporozoite-Induced and Blood-Induced Infections of Plasmodium fallax in Turkeys

Age of infection in days (number of birds used)

Brain

Heart

Liver

Kidney

Lung

Spleen

Sporozoite-induced 5-g (5) 10 (1) 14-20 (2)

+ (5) -

+ (5) + -

+ (5) -

+ (5) -

+ (5) -

+ (5) + -

-

+ (11” + (1)” + (5)

+ Cl)+ (lib + 03) +

Blood-induced 34 (6) 5-6 (10) 7 (2) S-11 (4) 12-17 (8) 19 (1)

+ (116 + (‘53 +

+ O)‘+ (ljb + (5) +

+ (1)” + (lib + (4) +

a All excerythrocytic stagea found in same bird. b Only one of the two was infected with excerytbrocytic

+

(l)b -

+ (5) -

stages.

stages in most of the organs. The chief difference between the bloodinduced infections of the two host-parasite combinations was the fact that in the chicken-gallinaceum relationship there was a definite predilection of the exoerythrocytic stages for the spleen whereas in the turkeyfallax relationship these stages were found in only 2 animals out of 22 during the first 11 days and were not confined to the spleen in either of these birds. P. fallax in chickens. There was a striking difference in the degree of blood infection resulting from the inoculation of sporozoites as compared with erythrocytic stages of this species into chickens. No visible parasitemia resulted from any of the sporozoite inoculations whereas very heavy parasitemia resulted from blood inoculations (Table IV). This parasitemia reached its highest level during the first 6 days following inoculation and usually persisted for about 18 days. Only 2 birds out of 17 receiving sporozoites mere found to have any exoerythrocytic stages in their organs (Table IV). The heart, kidney, liver, and spleen were found to be inhabited by these stages in one bird 5 days after inoculation. In another bird killed 16 days after inoculation exoerythrocytic parasites were found only in the spleen and these exhibited cytologic evidence of deterioration.

EXOERYTHROCYTIC

STAGES

OF AVIAN

151

PLASMODIA

Similarly only 2 birds out of 24 which were inoculated with erythrocytic stages were found to harbor exoerythrocytic stages-one of them was killed 6 days and the other 18 days after inoculation. Parasites were seen in the spleen only of each and in the latter they appeared to have undergone deterioration (Table IV). P. fullaz in other hosts. This species of parasite has been an especially valuable one for studying the differences in infections which depend upon differences in the host. Unlike P. gallinaceum, P. jallax has a very wide spectrum of infectivity for hosts. Besides the experiments just described on chickens and turkeys, four other gallinaceous birds and also ducklings, pigeons, and canaries were studied. A summary of the susceptibility of these birds as expressed in parasitemia and the occurrence of exoerythrocytic stages in the tissues of animals infected by means of sporozoites and of erythrocytic stages is given in Table VI. Of the 6 gallinaceous birds (including chickens and turkeys) studied some parasitemia resulted in each case from the inoculation of sporozoites. The degree of blood infection varied from slight to none in pheasants, through medium in quail, and medium to heavy in partridges to heavy in guinea fowl. No exoerythrocytic stages were found in sporoTABLE Parasitemia

VI

and Occurrence

Plasmodium

of Ezoerythrocytic fallax in Various Birds

Sporozoites inoculated

Host

Degree of Parasitemia

Duckling

Slight to none

Pigeon Canary

Slight to none Slight

Quail Pheasant Partridge Guinea fowl

Organs containing exoerythrwytic stages

None in 18

None in gb Heart of 1 in 3 (8 days) Medium Spleen of 1 in 8 (6 days) Slight to none None in 3b Moderate to heavy All principal organs of 4 in 8 (4, 5, 6 days) Heavy None

Stages of

Erythrocytic Degree of Pamsitemia

Medium

Heavy Medium to heavy Heavy Heavy

stages inoculated Organs containing exoerythrocytic stages

Spleen0 of 1 of 12 (12 days) None in 7 None in 9

None in 3

Light

o Evidence of degeneration. b Ckyptoeoitee were en in other birds given subcutaneous inoculations

of sporozoitea.

152

CLAY

G. HUFF

zoite-induced infections in guinea fowl and none was found in the pheasants which received intravenous inoculations of sporozoites, but cryptozoites were found in the local skin areas into which sporozoites had been inoculated. The spleen was found to harbor exorythrocytic stages in one out of eight sporozoite-induced infections in quail. Four out of eight partridges with sporozoite-induced infections had generalized organ infections on the 4th, 5th, and 6th days after inoculation. Ducklings, pigeons, and canaries were only slightly susceptible to sporozoites. All of the species tested (Table VI) by inoculations of infected blood exhibited some parasitemia but only one degenerated exoerythrocytic stage was found in the spleen of 1 duckling of 12 so inoculated and none in pigeons, canaries, and pheasants. P. lophurae in chickens and turkeys. Parasitemia was demonstrated in earlier experiments by Porter and Laird (1946) on chicks, ducklings, turkey poults, pheasant chicks, canaries, and guinea keets following inoculations of sporozoites. Unfortunately all strains of this parasite which were available at the time of the current work were incapable of infecting mosquitoes. Therefore, the tissues of only chicks and turkeys with blood-induced infections were searched for exoerythrocytic infection. Heavy parasitemias resulted from these inoculations in both kinds of host. No tissue stages were found in 9 chicks (Table IV). The distribution of exoerythrocytic stages in blood-induced infections in turkeys is shown in Table VII. It should be noted that there was no trace of the earlier invasion of spleen and some other organs characteristic of bloodinduced infections of P. gallinaceum and P. fallax in chickens and turkeys and a few other host-parasite combinations shown in Table IV. The similarity of the later invasion of all principal organs in this infection to that of P. jallax infections in turkeys will be discussedlater in this paper. TABLE Distribution Age of infection in days (number of birds used)

3-10 (15) 11-12 (7) 13-14 (3) 15 (1) 16-18 (2)

Brain

of Ezoerythrocytic of Plasmodium

Heart

VII

Stages of Blood-Induced lophurae in Turkeys

Kidney

-

-

-

-

+(l) + (3) + f(2)

-

-

-

+

(3) + -

+

(2) + -

Lung

Liver

+

Spleen

(2)

-

Infections

+ +

(1) (2)

-

Thyroid

+ +

-

-

(2) -

(1)

+

Illtestine

(1)

EXOERYTHROCYTIC

Distribution

Inoculum

of Ezoerythrocytic

STAGES

OF AVIAN

153

PLASMODIA

TABLE VIII Stages of Plasmodium cathemerium

Age of infection in days (number of birds used) Brain

lj$-2 (2) Sporozoites Sporozoites 4 (2) Sporozoites 5-6 (4) +-Cl) 4(3) Infected blood Infected blood 5(2) 6-18 (9) Infected blood

Heart

Kidney

Liver

Lung

-

-

-

-

-

I -

+-Cl) -

in Canaries

Spleen -

+(I) +-(2) + (1) + (4) + (1) + (1) + (1) -

Inkstine

BOW2 marrow

-

-

-

-

-

-

P. relictum in canaries and pigeons. This is the first species of avian malaria to be studied experimentally. Its behavior in canaries is well known. Parasitemia is easily produced by inoculation of either sporozoites or erythrocytic stages. As shown in Table IV only 1 canary of 6 inoculated with sporozoites and 1 of 14 inoculated with erythrocytic stages were found to harbor exoerythrocytic stages. Blood-induced infections of Coatney’s strain of P. relicturn were readily produced in pigeons. Although heavy blood infections resulted, only 2 pigeons of 10 were found to harbor exoerythrocytic stages. They were in the heart of a 12-day infection and the spleen, heart, and brain of a 15-day infection (see Table IV). P. cathemerium and P. circumflexurn in canaries. Although parasitemia in canaries was produced by the inoculation of infected blood in each of these species, exoerythrocytic stages of the former were found in the liver, lung, and spleen of only 1 bird of 14 of the former (Table VIII) and none was found in 16 infections of the latter (Table IV). Exoerythrocytic stages were comparatively more abundant in sporozoite-induced infections of P. cathemerium (Table VIII). P. elongatum in canaries and gosling embryos. The distribution of exoerythrocytic stages of this species in blood-induced infections in canaries and gosling embryos is shown in Table IX. Comparisons of the results from this species with those of other species of Plasmodium herein reported, as well as with the results of Huff and Bloom (1935) on the same species, will be made in the Discussion section. Comparisons of Erythrocytic and Exoerythrocytic Infections in Various Host-Parasite Combinaticms The frequency with which exoerythrocytic stages are encountered in the fixed tissues of the host has often been believed to be an expression

154

CLAY G. HUFF

TABLE IX Distribution

Host

Stages in Blood-Induced Infections of Plasmodium elongatum in Gosling Embryos and Canaries

of Ezoerythrocytic

Age of infection in days (number of birds used) Brain

Gosling

embryo Gosling embryo Canary Canary

Canary Canary l

4-5 (4)

-

6-7 (6)

-

3-5 (5) 6-8 (4) 9-12 (3) 13-16 (3)

-

Heart

Kidney

Liver

-

-

-

+

(2) + (1) + -

-

Lung

Bone marrow

(4) +

-

Spl.2Xl

+(I) -

(1) +

(3)* + (1)

+ cl)* + w* -

+ (2) + (3) + (2) -

In lymphocyte.

of the virulence of the infection in the host. To test this hypothesis all of the data from these studies in which there were some quantitative expressions of both the degree of parasitemia (at its peak) and the frequency of occurrence of exoerythrocytic stages were collected and subjected to a test for coefficient of correlation. Nineteen sets of such data were available and represented 5 examples of sporozoite-induced and 14 examples of blood-induced infections. Four examples were of P. gallinaceum, 9 of P. jallax, 2 of P. lophurae, 2 of P. relicturn and one each of P. cathemerium and P. circumjlexum infections. These data were only semiquantitative but they could easily be placed in an ordinal arrangement. The coefficient of correlation was found to be +0.14. DISCUSSION

Throughout the following discussion no consideration is given to the first 3 days of the infection. It is known from a considerable amount of published work (Porter, 1942; Huff and Coulston, 1944; Huff et al., 1947; Coulston and Huff, 1947; Huff, 1951) that cryptozoites of several of the avian malarial parasites occur in cells of the lymphoid-macrophage system either at the site of mosquito bite or in the organs of the infected animal. Furthermore it is known that exoerythrocytic stages developing subsequently invade the vascular endothelium and from that time forward, when found at all, they are nearly always found in these cells. Moreover, no exoerythrocytic stages of any of the avian malarias (with the possible exception of P. elongatum) are known during the first 3 days of infections produced by blood inoculation. Therefore, when the early

EXOERYTHROCYTIC

STAGES

OF AVIAN

TABLE

X

PLASMODIA

155

Summary of Patterns of Distribution of Exoerythrocytic Stages of P. gallinaceum and P. fallax in Chickens and Turkeys Host

Parasite

Chicken

P. gallinaceum

Turkey

P. jallax

Chicken

P. jallaz

Turkey

P. gallinaceum

Sporozoite-induced

Blood-induced

3-6 Generalized infection, 3- Spleen infection, days; (1 liver infec10 days; loss after 10 tion) ; generalized indays fection, IO-18 days Generalized infection, 5- Occasional occurrence in principal organs, 5-7 10 days; loss after 10 days; generalized indays fection, 12-19 days parasites in Sparse parasites in prin- Sparse spleen, 6th day cipal organs, 5th day Degenerate parasites in Sparse, degenerated parasites in spleen, 18th spleen, 16th day day Spleen, brain infected, Spleen only, 5-16 days 5-8 days Two other organs, 14-16 Generalized infection, days 12-16 days

portion of the infection is mentioned hereafter I shall be referring to the period approximately from the 3rd to the 9th days following inoculation. In attempting to analyze the characteristics of the distribution of the exoerythrocytic stages in the various parasite-host combinations it will be advantageous to consider first the infections of P. gallinaceum and P. fallax in chickens and turkeys. Complete series of both sporosoiteinduced and blood-induced infections were obtained of both species of parsites separately in both types of hosts. A summary of the principal characteristics of the infections of these eight combinations is presented in Table X. In general, the distribution of the exoerythrocytie stages tended to be divided into an early (3-9 days) and a late (lo-19 days) period regardless of the type of inoculum employed. The findings in P. gallinaceum infections in chickens closely paralleled those of P. fallax in turkeys and together they represent the type of exoerythrocytic infection encountered in examples of the most highly susceptible hosts. In this type of parasite-host relat.ionship sporozoite-induced infections were characterized by an early appearance of exoerythrocytic stages in most of the organs of the host. After 10 days these stages were rarely found. ,On bhe other hand, there was only occasional occurrence of exoeryth-

156

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rocytic stages in the early portion of blood-induced infections. When found at all they were most frequently encountered in the spleen but their numbers in this early portion of the infection were always far less than in sporozoite-induced infections. Following this sparse occurrence in the early portion of the infection, however, the exoerythrocytic stages became abundant and generalized in the latter portion of blood-induced infections. Animals which survived the early parasitemia resulting from bloodinduced infections often exhibited symptoms of involvement of the central nervous system with a high mortality. Post-mortem examination of the tissues of birds dying at this stage revealed large concentrations of exoerythrocytic stages in the vascular endothelial cells of all organs. Although death usually was the result of brain involvement it would probably have resulted from damage to other organs if the brain had not been involved. The late appearance of generalized infections in these two combinations of host and parasite are in agreement with the findings already reported on P. gallinaceum infections in chicks by Huff and Coulston (1946) and by Haas et al. (1948). There was no indication of an earlier invasion of the capillary endothelium of the brain than of other organs as observed by Lewert (1948) in blood-induced infections of P. gallinaceum in chicks which were treated with quinine. The converse combinations-P. gallinaceum in turkeys and P. fallax in chickens-were characterized by sparse exoerythrocytic stages in the early portion and even less frequent ones in the latter portion and this situation held in both sporozoite- and blood-induced infections. It is, perhaps, significant in both instances that the exoerythrocytic stages observed in the later stage exhibited evidence of deterioration. Such degenerative changes have been noted in P. lophurae infections in chickens, ducks, turkeys, and guinea fowl (Huff, Coulston, Laird, Porter, 1947); and in P. relicturn and P. gallinaceum infections in ducks (Huff, 1951). In those instances they were believed to have been due to the natural immunity of the host. However, similar degenerative changes were noted (Huff, 1952) in primary infections of P. gallinaceum in chicks, and hence were probably caused by some mechanism of acquired immunity. In the results here reported for blood-induced infections of P. fallax in chickens no evidence of degenerative change was seen in the exoerythrocytic stages in the early part of the infection. It is believed that these findings strengthen the belief that the action of acquired immunity of the host may be manifested by the appearance

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of the exoerythrocytic stages. In sporozoite-induced infections of P. gallinaceum in turkeys, sparsely occurring exoerythrocytic stages were observed during the early stage, but unlike the results of P. fallax in chickens, they, in the later stage, were more abundant and were normal in appearance. They did not exert so pathogenic an action on the turkey as the similar stages in this species in the chicken. Although the number and distribution of exoerythrocytic stages were similar in the early portion of blood-induced infections of P. gallinaceum in turkeys and P. fallax in chickens the former appeared to show more predilection for the spleen than the latter, thus resembling the findings in blood-induced infections of this species in chickens. The results in the later portion of the infections for the blood-induced infections of P. fallax in chickens and P. gallinaceum in turkeys were essentially similar except that in the latter (as in the case of sporozoite-induced infections) no degenerated forms were observed. This comparison of two species of parasites in two species of hosts reveals the importance of both the parasite and the host in establishing a pattern of occurrence and distribution of the exoerythrocytic stages. It also reveals that the combinations least favorable to the host are alike in pattern whether the infections were sporozoite- or blood-induced. Of all the parasites studied P. fallax had the widest range of infectivity of hosts. From the examination of the summaries in Tables VI and X it is apparent that the occurrence and distribution of exoerythrocytic stages in the nine hosts ranges from none to very abundant and generalized. Taking into account the degree of parasitemia as well as the characteristics of the exoerythrocytic stages in blood- and in sporozoiteinduced infections one may arrange the hosts approximately in the following order of susceptibility to P. fallax: turkey, partridge, chicken, quail, guinea fowl, pheasant, canary, pigeon, and duckling. This wide range of infectivity suggests a close relationship between P. fallax and P. pin&ii, isolated from a toucan in Brazil (Muniz and Soares, 1953). The describers of this species found it to be infectious to a wide range of hosts including pigeons, chickens, sparrows, canaries, ducks, turkeys and several indigenous Brazilian birds. They failed, however, to find any exoerythrocytic stages. I have examined blood smears of their new species, kindly sent me by Dr. Muniz, and have found P. pinottii to be morphologically distinct from P. fallax. The patterns of exoerythrocytic development of blood-induced infections of P. lophurae and P. fallax in turkeys were very similar (Tables V

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and VII). No exoerythrocytic stages were found in the early portion of the P. lophurae infection and in only two birds (of 20) of P. fallax were they seen. Moreover, they became abundant and generalized in the later portions of infection of both species. Turkeys infected by blood-induced P. lophurae exhibited symptoms of central nervous system involvement prior to death just as they did when infected with blood-induced P. fallax. This striking similarity of behavior of the exoerythrocytic stages of the two species in turkeys suggests a close zoological relationship between them. Manresa (1953) reported a similar distribution of exoerythrocytic stages of P. lophurae in blood-induced infections in turkeys. The development of a generalized exoerythrocytic infection in the later portion of blood-induced infections of P. lophurae and P. jallax in turkeys was exhibited also in blood-induced infections of P. gallinaceum in chickens and turkeys. There appeared to be remnants of such tendency also in blood-induced infections of P. jallax in ducklings and chicks and of P. relicturn in canaries and pigeons. The reason for the long delay in entrance into endothelium of the vasclar system even though it is accessible to the parasites from the beginning of parasitemia is still unknown. Previous studies (Huff, 1954) indicated a possible role of the splenic reticular cells in effecting changes in the parasites in reference to their ability to invade vascular endothelium. This hypothesis has been greatly weakened by the following facts: (1) unpublished work proving that late invasion of the vascular endothelium can occur in splenectomized chickens inoculated with erythrocytic stages of P. gallinaceum; (2) the finding of exoerythrocytic stages early in the infection in organs other than spleen in blood-induced infections of P. gallinaceum in chickens (Table II), turkeys (Table III) and pheasants (Table VI), P. cathemerium in canaries (Table VIII), and of P. jallax in turkeys (Table V); and (3) the absence of any exoerythrocytic stages in the early stage but generalized infection with them in the later stage of blood-induced infections of P. lophurae in turkeys (Table VII). The degree of parasitemia does not appear to be correlated to the numbers of exoerythrocytic stages from an over-all viewpoint. However, a quantitative study needs to be made upon one of the examples in which heavy exoerythrocytic infection results in blood-induced infections (e.g. P. jallax or P. lophurae in turkeys) to determine whether such a correlation exists. Certainly no obvious correlation between size of inoculum and degree of exoerythrocytic infection was found in the two such parasite-host combinations tested. In my results, as well as in those of Rossan (1957), the length of time

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elapsing between inoculation and the general appearance of exoerythrocytic stages in blood-induced infections appeared to be the one almost invariable condition. Manresa (1953) found no influence of sex, immune plasma, or intensity of parasitemia upon the development of exoerythrocytic stages of P. lophurae in blood-induced infections in turkeys. Attempts to analyze further the factors responsible for the acquisition of the ability by the parasite to invade vascular endothelium are in progress. In considering the distributions of exoerythrocytic stages in the various parasite-host combinations here reported it should be emphasized that differences between the results here recorded and the results from earlier work of mine and of other investigators may possibly be explained by variables other than those due to species of host and of parasite. For example, it is known that among morphologically identical strains of a given species great differences may exist in their capacities for producing exoerythrocytic stages in a particular species of host. The differences previously pointed out by Porter and Huff (1940) between the exoerythrocytic stages of P. elongatum and the other avian parasites and further discussed by Porter (1942) have been confirmed by the results of this study. With the exception of erythrocytes (and their immediate precursors) and macrophages, the preferences for cell type of P. elongatum and the other avian malarial parasites here considered are mutually exclusive (Huff and Bloom, 1935; Porter, 1942). The exoerythrocytic stages of P. elongatum occur in hemocytoblasts, granulocytes, thrombocytes, macrophages, monocytes, and plasma cells while those of the gallinaceum-type (which includes all others considered in this paper) occur in cells of the lymphoid-macrophage series and in vascular endothelium. Especial attention was paid in the present, series of sections of birds infected with P. elongatum to the reticular cells of the spleen which is the favored site of exoerythrocytic stages in bloodinduced infections of the other species of Plasmodium. All exoerythrocytic stages of P. elongatum seen in the spleen were in lymphocytes and in no instances were they seen in the splenic reticular cells or in the vascular endothelium of any of the organs. The gosling embryo was found to be a good host to this species and on the 6th day of infection exoerythrocytic stages were fairly well generalized in these embryos. Although further study of P. elongatum is needed before one can be confident, it now appears that none of the generalizations apply to the occurrence and distribution of its exoerythrocytic stages which apply to those with the gallinaceum-type. I have

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called attention to the similarities between this species and P. hugi (Huff, 1953) and the possibility that P. nucleophilum may also belong to this type (Huff, 1956). SUMMARY AND CONCLUSIONS The distribution of exoerythrocytic stages of seven species of avian malaria in the principal tissues of nine species of birds during the course of infection was determined. Of the 34 parasite-host combinations 15 were induced by sporozoites and 19 by blood. Infections in chickens and turkeys by P. gallinaceum and P. jallax respectively, which represented examples of the highest susceptibilities of hosts, were characterized as follows: (1) in sporosoite-induced infections exoerythrocytic stages were prevalent and widely distributed during the early portion of the infection (3-9 days) and disappeared in the late portion (lo-19 days) ; (2) in blood-induced infections exoerythrocytic stages were scarce during the early portion but prevalent and widely distributed in the latter portion of the infection. In the converse combinations (chickens and turkeys infected with P. jallax and P. gallinaceum respectively) the distribution of exoerythrocytic stages in sporozoite-induced infections of P. gallinaceum in turkeys was approximately like that of blood-induced infections mentioned under (2) above. In the three other combinations sparse, and sometimes degenerate, parasites were found in both early and late portions of the infection. Blood-induced infections of P. lophurae in turkeys resembled those similarly induced by P. jallax in turkeys. In both of these infections exoerythrocytic stages appeared with clocklike regularity on the 12th or 13th day following inoculation, were wide-spread in the tissues, and almost invariably resulted in the death of the host. In the following parasite-host combinations no exoerythrocytic stages were found even though in all cases parasitemia resulted: sporozoiteinduced gallinaceum infections in partridges, sporozoite- and blood-induced infections of jallax in pigeons, sporozoite- and blood-induced infections of jallax in pheasants, sporozoite-induced jallax in guinea fowl, blood-induced jallax in canaries, blood-induced lophurae in chicks and blood-induced circum$exum in canaries. In the remaining parasite-host combinations, representing the less susceptible hosts to the parasites concerned, exoerythrocytic stages were found only rarely, showed a preference for the spleen when they

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did occur, and were more often found in the early than in the late part of the infection. From these results the following generalizations can be made: (1) Exoerythrocytic stages (of the gallinaceum-type) are less likely to occur in blood-induced infections than in sporozoite-induced infections. (2) Exoerythrocytic stages occur earlier in infections induced by sporozoites than they do in infections induced by blood. (3) Definite patterns of exoerythrocytic occurrence and distribution are not characteristic of host speciesor of parasite species,but are determined by both. No clear relationship was established between degree of exoerythrocytic infection and (1) size of inoculum in blood-induced infections or (2) degree of parasitemia in infections produced by either inoculation of sporozoites or infected blood. In the casesin which the exoerythrocytic infection following inoculation of infected blood was severe and generalized the most important factor determining its onset was the length of the period of infection of the host. The types of host cells invaded by P. elongaium are so different from the ones occupied by all of the other specieshere considered as to make unlikely the application to it of any of the above generalizations. REFERENCES COULSTON, F., CANTRELL, W., AND HUFF, C. G. 1945. The distribution

and localization of sporozoites and pre-erythrocytic stages in infections with Plasmodium gallinaceum. J. Infectious Diseases 76, 226-238. COULSTON, F., AND HUFF, C. G. 1947. The morphology of cryptozoites and metacryptozoites of Plasmodium relicturn and the relationship of these stages to parasitemia in canaries and pigeons. J. Infectious Diseases 80, 209-217. HAAS, V. H., WILCOX, A., LAIRD, R. L., EWING, F. M., AND COLEMAN, N. 1948. Symposium on exoerythrocytic forms of parasites. VI. Response of Exoerythrocytic forms to alterations in the life-cycle of Plasmodium gallinaceum. J. Parasitol. 34, 306-320. HUFF, C. G. 1948. Natural immunity

and susceptibility of doves and pigeons to exoerythrocytic and erythrocytic stages of Plasmodium relictum. Proc. Znternat. Congr. Trap. Med. & Malaria, 4th Congr. Washington, D. C. 602-606. HUFF, C. G. 1951. Observations on the pre-erythrocytic stages of Plasmodium relictum, P. calhemerium and P. gallinaceum in various birds. J. Infectious Diseases 88, 17-26. HUFF, C. G. 1952. Studies on the exoerythrocytic stages of Plasmodium gallinaceum during the “transitional phase.” Exptl. Parasilol. 1, 392405.

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HUFF, C. G. 1953. Observations on Plasmodium Am. J. Trap. Med. and Hyg. 2, 62&623.

h@

Muniz, Soares, and Batista.

HUFF, C. G. 1954. Changes in host-cell preferences in maiarial parasites and their relation to splenic reticular cells. J. Infectious Diseases 94, 173-177. J. ParaHUFF, C. G. 1956. Exoerythrocytic stages of Plasmodium nucleophilum. sitol. 49, 612. HUFF, C. G. AND BLOOM, W. 1935. A malarial parasite infecting all blood and blood-forming cells of birds. J. Infectious Diseases 67, 315-336. HUFF, C. G., AND COULSTON, F. 1944. The development of Plasmodium gallinaceum from sporozoite to erythrocytic trophozoite. J. Infectious Diseases 76,231-249. HUFF, C. G., AND COULSTON, F. 1946. The relation of natural and acquired im-

munity of various avian hosts to the cryptozoites and metacryptozoites of Plasmodium gallinaceum and P. relictum. J. Infectious Diseases 78, 99-117. HUFF, C. G., COULSTON,F., LAIRD, R., AND PORTER, R. J. 1947. Pre-erythrocytie development of Plasmodium lophurae in various hosts. J. Infectious Diseases 81, 7-13. LEWERT, M. 1948. Exoerythrocytic infection by Plasmodium gallinaceum in bloodinduced, quinine treated chicks. Am. J. Hyg. 49, 158-170. MANRESA, M., JR., 1953. The occurrence of phanerozoites of Plasmodium lophurae in blood-inoculated turkeys. J. Parasitol. 39, l-4. MCGHEE, R. B, 1951. The adaptation of the avian malaria parasite Plasmodium lophurae to a continuous existence in infant mice. J. Infectious Diseases 88, 86-97. MUNIZ, J., AND SOARS, R. DE R. L. 1953. Nota s8bre urn parasita do gbnero Plasmodium eneontrado no Ramphastos taco Miiller, 1776, “Tucano-aFu,” e diferente do Plasmodium hu&: Plasmodium pinotlii, n. sp. 11th Congr. Brasil de Higiene, Cur&a, 1621 Nov. 1963: 1-9. PORTER, R. J. 1942. The tissue distribution of exoerythrocytic schizonts in sporozoite-induced infections with Plasmodium calhemerium. J. Infectious Diseases

71, 1-17. PORTER, R. J., AND HUFF, C. G. 1940. Review of the literature

on exoerythrocytic schizogony in certain malarial parasites and its relation to the schizogonic cycle in Plasmodium elongatum. Am. J. Trop. Med. 20, 869-888. PORTER, R. J., AND LAIRD, R. L. 1946. Unpublished data (See Huff, Coulston, Laird, and Porter, 1947 above). ROSSAN, R. N. 1957. The effect of antimalarial drugs on the exoerythrocytic and erythrocytic stages of blood-induced infections of Plasmodium fallax in the turkey. Exptl. Parasitol. 6, 163-188.