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Histomonas meleagridis and H. wenrichi: Time of acquisition by Heterakis gallinarum
EXPERIMENTAL
PARASITOLOGY
Histomonas
29,%-65
(1971)
meleagridis and H. wenrichi: by Heterakis gallinarum Everett
National
Time
of Acquisition
E. Lund
Animal Parasite Laboratory, Veterinary Sciences, Research Agricultural Research Service, U.S. Department of Agriculture Beltsville, Maryland XV05 (Submitted
for publication,
22 June
Division,*
1970)
LUND, EVERETT E. 1971. Histomonas meleagridis and H. wenrichi: Time of Acquisition by Heterakis gallinarum. Experimental Parasitology 29, 5995. Concurrent infection of young chickens with Histomonas-free Heterakis gallinarum less than 10 days old and Histomonas meleagridis, with or without H. wenrichi, did not result in the acquisition, retention, and subsequent transmission of either protozoan by the next generation of cecal worms. Concurrent infections of chicks or poults with Histomonas-free Heterakis more than 10 days old and either species of Histomonas did result in the acquisition, retention, and subsequent transmission of the protozoa by some of the heterakids of the next generation. In all instances, evidence of such transmission was sought by feeding female cecal worms with embryonated eggs from each source to susceptible test poults. These were then watched carefully for evidences of Histomonas infection, by repeated examination of cecal discharges and by thorough examination at necropsy. Four of nine chickens that harbored both the cecal worm and histomonad simultaneously produced worms with eggs that initiated infections with H. meleagtidis. One such infection was detected for each 473 embryonated eggs. Association of Heterakis of 10 days or older with Histomonas wenrichi resulted in the latter’s acquisition, retention, and transmission with comparable frequency. Apparently, acquisition of Htitomonas that results in its retention and transmission by Heterakis is largely, if not exclusively, by cecal worms more than 10 days old, and hence. lumen dwellers. This has implications for drug control programs. INDEX
Life
History
DESCRIPTORS:
; Transmission;
Histomonas Control
meleagridis, ; Chickens;
Heterakis
gallinarum;
the causal agent of blackhead. Lund (1968), using birds with experimentally produced simultaneous infections of H. wenrichi and Heterakis gallinarum, found that this protozoan was most likely to be acquired by the cecal worms when they are larvae 12-20 days old. Histomonas meleagridis was not present in the birds used for those studi,es. There is, however, one report of a similar study involving H. meleagridis. Niimi (1937) found what he regarded as
The common cecal worm of poultry, Heterakis gallinarum (Schrank, 1788) Madsen, 1949, is a vector for the nonpathogenic protozoan, Histomonas wenrichi (Lund, 1963) [=Parahistomonas wenrichi (Lund, 1963) Honigberg and Kuldova, 19691, as well as for the pathogenic protozoan Histomonas meleagridis (Smith, 1895) Tyzzer, 1920, * Formerly tory, Animal sion.
H. wenrichi; Turkeys.
Beltsville Parasitological LaboraDisease and Parasite Research Divi-
59
60
LUND
blackhead organisms eit,her in the intestine or ovary or both of several heterakids first exposed t,o t’he protozoa as the worms were 16-25 days old. In two instances, he was able to transmit the organism by feeding these \vorms. He made no egg counts, but some results of his study and ours can be compared. The experiments reported here were designed to determine the stages in its life history (bissue or lumen stages) when Heterakis acquires Histomonas and retains it so transmission by its eggs can be demonstrated. Such acquisition was to be tested both in the presence and the absence of H. wenrichi because both situations occur in nature. Sdditionally, these studies were designed to provide some means of judging the period of association of histomonads and heterakids other than relying solely on t,he det,ection of histomonads in the cecal discharges. This was to be accomplished by developing the worms to almost 10 days in one host (either infected with Histomonas or free of the parasite), then transferring t’he larvae to another host in which the opposite situation prevailed, and, finally, feeding eggs in worms developed in each of the above sit’uations to susceptible poults which were then checked for Histomonas. MATERIALS
AND
METHODS
Heterakis. The strain of Heterakis galliused for these studies was originally collected from turkeys raised on a farm that had apparently been free of blackhead for several years. None of 74 female worms transmitted Histomonas meleagridis, but at least two transmitted the nonpathogenic H. wenrichi (Lund, 1963). During the next 3 years, seven generations of cecal worms were grown in several hundred chickens and turkeys. H. meleagridis never appeared in the cecal discharges or cecal contents of any bird, nor did any bird ever show other evidences of histomoniasis. From birds that had not shown H. wenrichi either, we then selected worms to be free of it also, and narum
with the next three generations of worms we found no evidence of transmission of this histomonad. For the last of the three passages we used more t,han 17,000 embryonated eggs, pooled to increase chances of infection (Lund and Burtner, 1957)) for inoculating 27 chicks and 6 poults. Finding no histomonads in any of these birds eit’her, we considered their heterakids to be “Histomonas-free.” There had now been at least 10 generations of worms with no association with H. meleagridis. Histomonas. The strain of H. wenrichi used in these studies was that isolated and described previously (Lund, 1963)) which had been maintained in chickens and turkeys at the National Animal Parasite Laboratory for several years. The &rain of H. meleagridis was one also maintained in chickens and turkeys at this Laboratory for many years. It had consistently produced high mortality in turkeys, but only transitory cecal pathology in crossbred chickens from sources to be used in these experiments (Lund, 1967). Birds. The chickens used for these studies were a first-generation cross of New Hampshire males and Barred Plymouth Rock females. The turkeys were Beltsville Small Whites. On all tests, the birds were 4-6 weeks old at the time of their first inoculation. They were caged individually in each experiment, thus enabling identification of their cecal discharges. Acquisition of Histomonas by Heterakis larv.ae lessthan 10 days old. At midmorning of the day the test started (after the birds had evacuated their ceca), each of 10 poults received rectally 1 ml of a saline suspension containing about 50,000 Histomonas wenrichi. The following morning each bird again received 50,000 H. wenrichi, but this time 1000 H. meleagridis were also present. That afternoon (when all birds had had ample time to feed), each poult received orally by pipette to the stomach approximately 360 embryonated Heterakis eggs. The cecal discharges of each of t,hese 10
ACQUISITION OF Histomonas BY Heterakis
61
poults were then examined microscopically chickens had been given the Histomonasfor histomonads each day on days 4-9 after free Heterakis eggs, the cecal discharges of inoculation. Early on day 10, each poult each bird were examined daily for Histowas necropsied and all heterakids (then ap- monas. On day 10, each of nine chickens proaching 10 days of age) were removed, was necropsied, its Heterakis larvae rewashed in six changes of physiologic saline, moved, quickly washed in six changes of and transferred to a recipient parasite-free warm physiologic saline, and given rectally poult by rectal inoculation (Tyzzer and to one of the nine chicks that had received Collier, 1925). In one instance the hetera- Histomonas meleagridis 10 days before. In kids from the two ceca were given to sepa- like manner, the larvae from one of the rerate birds, thus making a total of 11 recipi- maining nine chicks were given to each of ent poults. the nine poults that had received H. wenriEach day for 27 days all available cecal chi 10 days before. During the next 28 discharges of these 11 poults were examined days, the cecal discharges of these nine for histomonads. On day 28 after the transchicks and nine poults, now foster hosts for fer of the larvae, the poults were necrop- the heterakids, were examined microscopisied, and all heterakids from each bird were cally for Histomonas. On day 28, each bird recovered and washed. The female worms was necropsied and examined for ,evidence were kept in 0.5% formalin for 3 weeks at of Histomonas infection. All Heterakis were room temperature (70-75” F) to permit the removed from each bird, and the females eggs to embryonate. These females were kept 3 weeks at room temperature in 0.5% then given orally, one worm to each bird, to formalin to permit embryonation of eggs. test poults whose cecal discharges were also The number of such eggs in each female examined for histomonads each weekday was counted, and two to seven females from until the birds were necropsied for study each chick or poult were given orally to a day 18 after the worms were fed. parasite-free poult. The cecal discharges of Concurrently with the above, each of 10 this bird were examined each weekday for poults received the saline suspension with Histomonas. On day 17, it was necropsied approximately 360 embryonated eggs from and examined for evidence of Histomonas the Histomonas-free Heterakis, but no His- infection. tomonas. Inasmuch as the Histomonas-free HeterAcquisition of Histomonas by stage-4 alcis were being maintained for source maHeterakis larvae, juveniles, or young terial, frequently by passage through both adults. In the morning, after the birds had chickens and turkeys, such birds, by rehad ample time to feed without disturbance, maining free of Histomonas, served as coneach of 18 parasite-free crossbred chickens stant controls. Their cecal discharges were was given 1 ml of physiologic saline con- examined for Histomonas frequently, and taining approximately 360 embryonated they too were necropsied for recovery of eggs from Histomonas-free Heterakis. The worms, which, in turn, were fed to new same morning, each of nine other parasite- groups of birds subjected to the same procefree crossbred chickens was inoculated rec- dures. tally with about 1 million Histomonas meRESULTS Zeagridis from liver lesions of a turkey. In Acquisition of Histomonas by Heterakis like manner, each of nine parasite-free larvae less than 10 days old. The results of poults received about 100,000 H. wenrichi this test are shown in Table I. Four of ten from the cecal discharges of an infected turpoults acquired infections with Histomonas key. From day 5 through day 9 after the 18 meleagridis, five developed infections with
62
LUND
TABLE E$ects
of Z’ransjerring Either Histomonas
1 Donor poult Infections established Histomonas meleagridis H. wenrichi No. of Heterakis larvae Recipient poult No. of examinatzions of cecal discharges (All were negative) No. of Heterakis recovered X0. of females a Left
and
right
I
lo-Da!/ Larvae of Heterakis meleagridis or H. wenrichi 2
3
gallinarum or Zioth 6
from Pozc!ts to Patyzsife-Free 7R”
7P
8
Infected Potrlts
with
9
10
Total
4 5 199
4
5
+ 18
25
25
18
20
42
+ 16:
+ + 13
lG
3
+ 3
24
25
23
22
22
24
22
25
25
25
25
2G2
0
0
0
0
0
0
0
A 3
8 5
3 0
0
17 8
+ +
+
+
ceca.
H. wenrichi, one bird had both, and two had neither. All 10 poults had some Heteral&. hfter the transfer of these larvae to 11 parasite-free poults (the 43 larvae from donor No. 7 having been given to two recipients), neither species of Histomonas was detected in any of the 262 examinations of cecal discharges made over the 27-day period before necropsy. Neither were they detected in any of the smears examined from the 22 ceca removed at necropsy. The 17 Heterakis that survived the 28 days after transfer were all in three poults that had received larvae from donors with Histomonas meleagridis. Of these 17 worms, 8 were females, rather small for 38 days, but at least 6 of them had eggs that were clearly embryonated. However, when these worms were fed to test poults, there was no evidence of the transmission of Histomonas. Acquisition of Histomonas by stage 4 larvae, juveniles, or young adult Heterakis. The results of the older heterakids’ acquisition of Histomonas meleagridis, as evidenced by the transmission of the protozoan to susceptible poults, are shown in Table II. The nine Histomonas-free donor chickens yielded from 13 to 106 Heterakis larvae, for a total of 420. Of the nine chickens inoculated with Histomonas meleagridis, only
one (No. 7) had voided the protozoan in sufficient numbers to be detectable in its cecal discharges, and in only one additional bird (iSo. 8) was H. meleagridis de&ted at necropsy. Bird No. 7 had no heterakids at’ necropsy, but the other eight recipient chickens had a total of 100 cecal worms. Of these, 37 were females in which 2366 embryonated eggswere later identified. Of the nine parasite-free poults that received the 37 female worms, two to seven per poult, one developed fatal histomoniasis and four ot’hers had subclinical infections. One recipient chicken (No. 4) provided the female heterakids given to t’wo poults that developed infections with Histomonas meleagridis. To sum up the results, three of the nine recipient chickens given H. meleagridis rectally 10 days before they became foster host’s to the lo-day-old Heteralcis larvae, matured at least one heterakid with at least one egg responsible for transmitting Histomonas meleagridis. The recipient chickens were Nos. 1, 6, and 9. One recipient chicken, No. 4, matured at least two such worms. Thus at least 5 of the 37 females fed to the test poults each had at least one egg that transmitted a viable histomonad. The results of that aspect of the study
ACQUISITION
Histomonas
OF
TABLE Transmission
Heterakis
BY
II
of Histomonas meleagridis with the Protozoan Only 1
by Eggs of Heterakis After the Larvae Were - - 2 3 4 5
21
24
13
106
9 3
4 1
3 1
34 5
270
90
100
-
Histomonas-free donor chicken No. of lo-day Heterakis Recipient chicken, given H. meleagridis 10 days before H. meleagridis detected No. of 38-day Heterakis No. of 38.day-females No. of embryonated eggs therein
63
-
gallinarum 10 or More
from Days
Worms Old
Associated
6
7
8
39
44
40
39
18
5
+ 0 0
+
G 3
8 4
18 7
490
320
-
130
530
4a
4b
8
9
2
4
4
96
210 ~__
130
5
6a
6b
Total
420
2366
Poult
that received these females and eggs Times cecal discharges examined Times histomonads detected (1 This
poult
died
20 days
25 0 infection
after
25 1
with
28 19 29 28 28 0 0 2 0 1 -I - severe lesions in both the ceca and the liver.
TABLE Transmission
of Histomonas
with
wenrichi the Protozoan Only
- -
Hislomonas-free donor chicken No. lo-day Heterakis transferred Recipient poult given H. wenrichi 10 days before H. wenrichi detected No. 38.day Heterakis No. 38-day females
- -
1
2
8
58
3a ~~__ poult receiving female Heterakis No. females fed No. of embryonated eggs in these females Times discecal charges examined Times H. wenrichi was detected
29 1
III
by Eggs of Heterakis gallinarum After the Larvae Were 10 or More - 3 4 5 6
117
75
79
46
+ 51 22
+ 33 14
0
+ 24 10
3b
5 poults
3c
4a
-
4b
6a
from Days
-
Associated
Worms
Old
- - - -
7
6
9
6
30
425
+ 12 6
7 129 55
+ 2 2 6b
7
-
8
--
-
9
Total
9 poults
Test
-
-
7
5
5
5
520
720
570
790
600
27
27
29
27
26
1
0
1
2
7
-
7
-
24
-
-
0 -
-i
2
6
47
320
1
540
4201
24
23
27
0
1 -
1
-
-
-
6 poults
-
64
LUND
that associated Histomonas wenrichi with Heterakis larvae older than 10 days are shown in Table III. A total of 425 heterakid larvae were recovered from the 9 Histomonas-free donor chickens, which had from 6 to 117 larvae each. Seven of the nine poults that received t’hese larvae also had Histomonas wenrichi while harboring the heterakids. Two poults (Nos. 2 and 5) had no Heterakis at necropsy, a third had no females, while a fourth poult had only one female which was discarded because it was alone in one cecum. The remaining five poults, all of which had Histomonas wenrichi, had 54 female heterakids, in 47 of which 4201 embryonated eggs were later identified. These 47 worms were fed to nine parasite-free poults, each of which received two to seven such worms, all from the same bird. Subsequently, six of these poults developed infections with H. wenrichi. Therefore, at least 6 of t’he 47 female worms must have had at least one egg that carried the histomonad. Four of the nine recipient poults that received Histomonas wenrichi and Histomonas-free Heterakis larvae, separately, were hosts to worms with such eggs. Two recipient’ poults (Nos. 3 and 4) had at least two such worms. DISCUSSION
AND
CONCLUSIONS
The results of the test that exposed Hetera&s larvae less than 10 days old to Histomonas are not sufficient to warrant a conclusion that such larvae never acquire histomonads and retain them so they are transmitted through the Heterakis eggs. Of the many larvae so exposed (at least 158), only eight were females that survived to maturity. None of these carried eggs that initiated a Histomonas infection in test poults. There are evidences from other sources that acquisition and retention by these young larvae may be quite uncommon. Among those investigators who reported having observed Histomonas in Heterakis, only Kendall (1959) found them in larvae
younger than 10 days. Two reviews of such findings have been published recently (Lee, 1969; Lund, 1969), the latter of which points out that Kendall’s finding represents a different situation, not indicative of recent acquisition. One explanation for the failure of young heterakids to acquire Histomonas wenrichi has been given elsewhere (Lund, 1968). Briefly, this protozoan is almost absent in the tissues, and not very abundant in the cecal lumen until more than 10 clays after its introduction. However, H. meleagridis is often fairly abundant in the lesions of t’he cecal wall 6 or 7 days after infection, and some of the Heterakis larvae are still there. By day 10, almost all of the larvae are in the lumen of the cecum, and H. meleagridis is also there, and sometimes quite numerous, largely because of extrusion from t’he lesions. Nevertheless, there are circumstanccs that tend to make acquisitions less frequent at this early time than later. The smaller, stage 3 larvae probably ingest less material than they will lat’er! and the histomonads are not so abundant in the cecal contents as they will be later. Furt’hermore, t’o be transmit’ted by the Heterakis eggs, the histomonads must persist in t’he worms until the latter mature. Both Siimi (1937) and Lee (1969) appeared to suggest that histomonads that survive long after being ingested have sought out a tissue that is hardly differentiated in Heterakis until Iv-e11 after day 10, the future ovary. Indeed, Siimi believed that histomonads in male worms merely disappear. If they do, presumably it is because they find no tissue appropriate for their preservation. Gibbs (1962) found H. meleagridis in many of the organs of mature male Heterakis. These observations can be harmonized by assuming that as male worms mature, they too differentiate tissues appropriate for the preservation of the histomonads. In contrast to the precarious courses described above for young larvae, the older larvae, juveniles, and young adult hetera-
ACQUISITIOE OF Histomonas kids are frequently afforded fairly good opportunities for acquiring H. meleagridis, and retaining them so that transmission by the embryonated eggs may occur. In this study, four of nine chickens in which Histo,monas-free Heterakis and Histomonas meleagridis were brought together produced worms with eggs that transmitted H. meleagridis. Niimi had only two such instances in 84 chickens. He fed 55 female Heteralcis to seven test chicks to get these two positives. In our test, 37 females with a total of 2366 embryonated eggs were given to nine poults, five of which developed infections with H. meleagridis. Thus, at least five of the 37 females must have had eggs that embryonated and transmitted H. meleagridis that retained viability sufficient to initiate detectable infections. For H. wenrichi the degree of transmission was comparable. Six of nine poults in which Histomonas-free Heterakis and Histomonas wenrichi were associated were later found to have heterakids with eggs that transmitted this protozoan. The six of 47 female worms carrying such eggs represent a ratio almost identical to that for worms carrying H. meleagtidis (five of 37). The results of these studies substantiate the conclusion drawn from the earlier test on acquisition (Lund, 1968) : that single simultaneous infections with Heterakis and Histomonas are entirely adequate to assure the perpetuation of the parasites, but the incidence of infection with Histomonas is low in both the definitive host and the intermediate host. To those interested in the purposeful control of histomoniasis, these circumstances suggest at least the following : 1. The complete exclusion of Histomonas from birds in restricted areas may be attainable by employing biological control through the use of appropriate management. 2. If drug control is to be used primarily or exclusively to prevent or reduce contaimination of soil with HGtomonas-bearing
RY Heterakis
65
eggs of Heterakis, an antihistomonal drug that is effect’ivc only in the lumen of the cecum may be adequate. This could bc a drug of low solubility, so that its presence in the tissues or elimination through the kidneys is inconsequential. Obviously, such a drug would not be selected for use with a species or breed of bird that was so susccptible to histomoniasis as to require that proliferation of the protozoa in the tissues bc halted by use of the drug.
REFERENCES B. J. 1962. The occurrence of the protozoan parasite Histomonns meleclgridis in the adults and eggs of the cecal worm Heternkis gallinae.
GIBBS,
Jourtlal HONGBERG,
of Protozoozogy
9,288-293.
B. M., AXD KULDOVA, J. 1969. Structure of a non-pathogenic histomonad from the cecum of galliform birds and revision of the trichomonad family Monocercomonadidae Kirhg. Journal of Protozoology 16,526535. KIGXDALL, S. B. 1959. The occurrence of Histomo~~us meleagridis in Heternkis gallinae. Pnrnsitology 49,16%172.
1~~.
D. L. 1969. The structure and development of Histomonas melengridis (Mastigamoehidae : Protozoa) in the female reproductive tract of its intermediate host, Heterakis gnllinarum (Kematoda). Pnmsitologv 59, 877-884. J,UND, E. E. 1963. Histomorzas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a nonpathogenic parasite of gallinaceous birds. Jozmnl of Protozoology 10,401-404. I,r:su, E. E. 1967. Response of four breeds of chickens and one breed of turkeys to experimental Heternkti and Histomonns infections. Avtin Diseases 11, 491-502. LUKD. E. E. 1968. Acquisition and liberation of H&omonns wenrichi by Heterakis gnllinnrum. Experimental Parasitology 22, 62-67. I,ITXD. E. E. 1969. Histomoniasis. Advances in Veterinrrry Science and Comparative Medicine 13,35&390. LUND. E. E.. AND BURTNER, R. H., JR. 1957. Infectivity of Hr terakis gallinne eggs with Histomonns mele~rgridis. Experimental Parasitology 6, 189-193. NIIYI. D. 1937. Studies of blackhead. II. Mode of Infection. Journal of the Japanese Society of Veterinargl Science 16, 183-239. TYZZER, E. E., AXD COLLIER, J. 1925. Induced and natural transmission of blackhead in the ahsence of Zieterakis. Journal of Infectious DCeases 31,265-276.
PARASITOLOGY
Histomonas
29,%-65
(1971)
meleagridis and H. wenrichi: by Heterakis gallinarum Everett
National
Time
of Acquisition
E. Lund
Animal Parasite Laboratory, Veterinary Sciences, Research Agricultural Research Service, U.S. Department of Agriculture Beltsville, Maryland XV05 (Submitted
for publication,
22 June
Division,*
1970)
LUND, EVERETT E. 1971. Histomonas meleagridis and H. wenrichi: Time of Acquisition by Heterakis gallinarum. Experimental Parasitology 29, 5995. Concurrent infection of young chickens with Histomonas-free Heterakis gallinarum less than 10 days old and Histomonas meleagridis, with or without H. wenrichi, did not result in the acquisition, retention, and subsequent transmission of either protozoan by the next generation of cecal worms. Concurrent infections of chicks or poults with Histomonas-free Heterakis more than 10 days old and either species of Histomonas did result in the acquisition, retention, and subsequent transmission of the protozoa by some of the heterakids of the next generation. In all instances, evidence of such transmission was sought by feeding female cecal worms with embryonated eggs from each source to susceptible test poults. These were then watched carefully for evidences of Histomonas infection, by repeated examination of cecal discharges and by thorough examination at necropsy. Four of nine chickens that harbored both the cecal worm and histomonad simultaneously produced worms with eggs that initiated infections with H. meleagtidis. One such infection was detected for each 473 embryonated eggs. Association of Heterakis of 10 days or older with Histomonas wenrichi resulted in the latter’s acquisition, retention, and transmission with comparable frequency. Apparently, acquisition of Htitomonas that results in its retention and transmission by Heterakis is largely, if not exclusively, by cecal worms more than 10 days old, and hence. lumen dwellers. This has implications for drug control programs. INDEX
Life
History
DESCRIPTORS:
; Transmission;
Histomonas Control
meleagridis, ; Chickens;
Heterakis
gallinarum;
the causal agent of blackhead. Lund (1968), using birds with experimentally produced simultaneous infections of H. wenrichi and Heterakis gallinarum, found that this protozoan was most likely to be acquired by the cecal worms when they are larvae 12-20 days old. Histomonas meleagridis was not present in the birds used for those studi,es. There is, however, one report of a similar study involving H. meleagridis. Niimi (1937) found what he regarded as
The common cecal worm of poultry, Heterakis gallinarum (Schrank, 1788) Madsen, 1949, is a vector for the nonpathogenic protozoan, Histomonas wenrichi (Lund, 1963) [=Parahistomonas wenrichi (Lund, 1963) Honigberg and Kuldova, 19691, as well as for the pathogenic protozoan Histomonas meleagridis (Smith, 1895) Tyzzer, 1920, * Formerly tory, Animal sion.
H. wenrichi; Turkeys.
Beltsville Parasitological LaboraDisease and Parasite Research Divi-
59
60
LUND
blackhead organisms eit,her in the intestine or ovary or both of several heterakids first exposed t,o t’he protozoa as the worms were 16-25 days old. In two instances, he was able to transmit the organism by feeding these \vorms. He made no egg counts, but some results of his study and ours can be compared. The experiments reported here were designed to determine the stages in its life history (bissue or lumen stages) when Heterakis acquires Histomonas and retains it so transmission by its eggs can be demonstrated. Such acquisition was to be tested both in the presence and the absence of H. wenrichi because both situations occur in nature. Sdditionally, these studies were designed to provide some means of judging the period of association of histomonads and heterakids other than relying solely on t,he det,ection of histomonads in the cecal discharges. This was to be accomplished by developing the worms to almost 10 days in one host (either infected with Histomonas or free of the parasite), then transferring t’he larvae to another host in which the opposite situation prevailed, and, finally, feeding eggs in worms developed in each of the above sit’uations to susceptible poults which were then checked for Histomonas. MATERIALS
AND
METHODS
Heterakis. The strain of Heterakis galliused for these studies was originally collected from turkeys raised on a farm that had apparently been free of blackhead for several years. None of 74 female worms transmitted Histomonas meleagridis, but at least two transmitted the nonpathogenic H. wenrichi (Lund, 1963). During the next 3 years, seven generations of cecal worms were grown in several hundred chickens and turkeys. H. meleagridis never appeared in the cecal discharges or cecal contents of any bird, nor did any bird ever show other evidences of histomoniasis. From birds that had not shown H. wenrichi either, we then selected worms to be free of it also, and narum
with the next three generations of worms we found no evidence of transmission of this histomonad. For the last of the three passages we used more t,han 17,000 embryonated eggs, pooled to increase chances of infection (Lund and Burtner, 1957)) for inoculating 27 chicks and 6 poults. Finding no histomonads in any of these birds eit’her, we considered their heterakids to be “Histomonas-free.” There had now been at least 10 generations of worms with no association with H. meleagridis. Histomonas. The strain of H. wenrichi used in these studies was that isolated and described previously (Lund, 1963)) which had been maintained in chickens and turkeys at the National Animal Parasite Laboratory for several years. The &rain of H. meleagridis was one also maintained in chickens and turkeys at this Laboratory for many years. It had consistently produced high mortality in turkeys, but only transitory cecal pathology in crossbred chickens from sources to be used in these experiments (Lund, 1967). Birds. The chickens used for these studies were a first-generation cross of New Hampshire males and Barred Plymouth Rock females. The turkeys were Beltsville Small Whites. On all tests, the birds were 4-6 weeks old at the time of their first inoculation. They were caged individually in each experiment, thus enabling identification of their cecal discharges. Acquisition of Histomonas by Heterakis larv.ae lessthan 10 days old. At midmorning of the day the test started (after the birds had evacuated their ceca), each of 10 poults received rectally 1 ml of a saline suspension containing about 50,000 Histomonas wenrichi. The following morning each bird again received 50,000 H. wenrichi, but this time 1000 H. meleagridis were also present. That afternoon (when all birds had had ample time to feed), each poult received orally by pipette to the stomach approximately 360 embryonated Heterakis eggs. The cecal discharges of each of t,hese 10
ACQUISITION OF Histomonas BY Heterakis
61
poults were then examined microscopically chickens had been given the Histomonasfor histomonads each day on days 4-9 after free Heterakis eggs, the cecal discharges of inoculation. Early on day 10, each poult each bird were examined daily for Histowas necropsied and all heterakids (then ap- monas. On day 10, each of nine chickens proaching 10 days of age) were removed, was necropsied, its Heterakis larvae rewashed in six changes of physiologic saline, moved, quickly washed in six changes of and transferred to a recipient parasite-free warm physiologic saline, and given rectally poult by rectal inoculation (Tyzzer and to one of the nine chicks that had received Collier, 1925). In one instance the hetera- Histomonas meleagridis 10 days before. In kids from the two ceca were given to sepa- like manner, the larvae from one of the rerate birds, thus making a total of 11 recipi- maining nine chicks were given to each of ent poults. the nine poults that had received H. wenriEach day for 27 days all available cecal chi 10 days before. During the next 28 discharges of these 11 poults were examined days, the cecal discharges of these nine for histomonads. On day 28 after the transchicks and nine poults, now foster hosts for fer of the larvae, the poults were necrop- the heterakids, were examined microscopisied, and all heterakids from each bird were cally for Histomonas. On day 28, each bird recovered and washed. The female worms was necropsied and examined for ,evidence were kept in 0.5% formalin for 3 weeks at of Histomonas infection. All Heterakis were room temperature (70-75” F) to permit the removed from each bird, and the females eggs to embryonate. These females were kept 3 weeks at room temperature in 0.5% then given orally, one worm to each bird, to formalin to permit embryonation of eggs. test poults whose cecal discharges were also The number of such eggs in each female examined for histomonads each weekday was counted, and two to seven females from until the birds were necropsied for study each chick or poult were given orally to a day 18 after the worms were fed. parasite-free poult. The cecal discharges of Concurrently with the above, each of 10 this bird were examined each weekday for poults received the saline suspension with Histomonas. On day 17, it was necropsied approximately 360 embryonated eggs from and examined for evidence of Histomonas the Histomonas-free Heterakis, but no His- infection. tomonas. Inasmuch as the Histomonas-free HeterAcquisition of Histomonas by stage-4 alcis were being maintained for source maHeterakis larvae, juveniles, or young terial, frequently by passage through both adults. In the morning, after the birds had chickens and turkeys, such birds, by rehad ample time to feed without disturbance, maining free of Histomonas, served as coneach of 18 parasite-free crossbred chickens stant controls. Their cecal discharges were was given 1 ml of physiologic saline con- examined for Histomonas frequently, and taining approximately 360 embryonated they too were necropsied for recovery of eggs from Histomonas-free Heterakis. The worms, which, in turn, were fed to new same morning, each of nine other parasite- groups of birds subjected to the same procefree crossbred chickens was inoculated rec- dures. tally with about 1 million Histomonas meRESULTS Zeagridis from liver lesions of a turkey. In Acquisition of Histomonas by Heterakis like manner, each of nine parasite-free larvae less than 10 days old. The results of poults received about 100,000 H. wenrichi this test are shown in Table I. Four of ten from the cecal discharges of an infected turpoults acquired infections with Histomonas key. From day 5 through day 9 after the 18 meleagridis, five developed infections with
62
LUND
TABLE E$ects
of Z’ransjerring Either Histomonas
1 Donor poult Infections established Histomonas meleagridis H. wenrichi No. of Heterakis larvae Recipient poult No. of examinatzions of cecal discharges (All were negative) No. of Heterakis recovered X0. of females a Left
and
right
I
lo-Da!/ Larvae of Heterakis meleagridis or H. wenrichi 2
3
gallinarum or Zioth 6
from Pozc!ts to Patyzsife-Free 7R”
7P
8
Infected Potrlts
with
9
10
Total
4 5 199
4
5
+ 18
25
25
18
20
42
+ 16:
+ + 13
lG
3
+ 3
24
25
23
22
22
24
22
25
25
25
25
2G2
0
0
0
0
0
0
0
A 3
8 5
3 0
0
17 8
+ +
+
+
ceca.
H. wenrichi, one bird had both, and two had neither. All 10 poults had some Heteral&. hfter the transfer of these larvae to 11 parasite-free poults (the 43 larvae from donor No. 7 having been given to two recipients), neither species of Histomonas was detected in any of the 262 examinations of cecal discharges made over the 27-day period before necropsy. Neither were they detected in any of the smears examined from the 22 ceca removed at necropsy. The 17 Heterakis that survived the 28 days after transfer were all in three poults that had received larvae from donors with Histomonas meleagridis. Of these 17 worms, 8 were females, rather small for 38 days, but at least 6 of them had eggs that were clearly embryonated. However, when these worms were fed to test poults, there was no evidence of the transmission of Histomonas. Acquisition of Histomonas by stage 4 larvae, juveniles, or young adult Heterakis. The results of the older heterakids’ acquisition of Histomonas meleagridis, as evidenced by the transmission of the protozoan to susceptible poults, are shown in Table II. The nine Histomonas-free donor chickens yielded from 13 to 106 Heterakis larvae, for a total of 420. Of the nine chickens inoculated with Histomonas meleagridis, only
one (No. 7) had voided the protozoan in sufficient numbers to be detectable in its cecal discharges, and in only one additional bird (iSo. 8) was H. meleagridis de&ted at necropsy. Bird No. 7 had no heterakids at’ necropsy, but the other eight recipient chickens had a total of 100 cecal worms. Of these, 37 were females in which 2366 embryonated eggswere later identified. Of the nine parasite-free poults that received the 37 female worms, two to seven per poult, one developed fatal histomoniasis and four ot’hers had subclinical infections. One recipient chicken (No. 4) provided the female heterakids given to t’wo poults that developed infections with Histomonas meleagridis. To sum up the results, three of the nine recipient chickens given H. meleagridis rectally 10 days before they became foster host’s to the lo-day-old Heteralcis larvae, matured at least one heterakid with at least one egg responsible for transmitting Histomonas meleagridis. The recipient chickens were Nos. 1, 6, and 9. One recipient chicken, No. 4, matured at least two such worms. Thus at least 5 of the 37 females fed to the test poults each had at least one egg that transmitted a viable histomonad. The results of that aspect of the study
ACQUISITION
Histomonas
OF
TABLE Transmission
Heterakis
BY
II
of Histomonas meleagridis with the Protozoan Only 1
by Eggs of Heterakis After the Larvae Were - - 2 3 4 5
21
24
13
106
9 3
4 1
3 1
34 5
270
90
100
-
Histomonas-free donor chicken No. of lo-day Heterakis Recipient chicken, given H. meleagridis 10 days before H. meleagridis detected No. of 38-day Heterakis No. of 38.day-females No. of embryonated eggs therein
63
-
gallinarum 10 or More
from Days
Worms Old
Associated
6
7
8
39
44
40
39
18
5
+ 0 0
+
G 3
8 4
18 7
490
320
-
130
530
4a
4b
8
9
2
4
4
96
210 ~__
130
5
6a
6b
Total
420
2366
Poult
that received these females and eggs Times cecal discharges examined Times histomonads detected (1 This
poult
died
20 days
25 0 infection
after
25 1
with
28 19 29 28 28 0 0 2 0 1 -I - severe lesions in both the ceca and the liver.
TABLE Transmission
of Histomonas
with
wenrichi the Protozoan Only
- -
Hislomonas-free donor chicken No. lo-day Heterakis transferred Recipient poult given H. wenrichi 10 days before H. wenrichi detected No. 38.day Heterakis No. 38-day females
- -
1
2
8
58
3a ~~__ poult receiving female Heterakis No. females fed No. of embryonated eggs in these females Times discecal charges examined Times H. wenrichi was detected
29 1
III
by Eggs of Heterakis gallinarum After the Larvae Were 10 or More - 3 4 5 6
117
75
79
46
+ 51 22
+ 33 14
0
+ 24 10
3b
5 poults
3c
4a
-
4b
6a
from Days
-
Associated
Worms
Old
- - - -
7
6
9
6
30
425
+ 12 6
7 129 55
+ 2 2 6b
7
-
8
--
-
9
Total
9 poults
Test
-
-
7
5
5
5
520
720
570
790
600
27
27
29
27
26
1
0
1
2
7
-
7
-
24
-
-
0 -
-i
2
6
47
320
1
540
4201
24
23
27
0
1 -
1
-
-
-
6 poults
-
64
LUND
that associated Histomonas wenrichi with Heterakis larvae older than 10 days are shown in Table III. A total of 425 heterakid larvae were recovered from the 9 Histomonas-free donor chickens, which had from 6 to 117 larvae each. Seven of the nine poults that received t’hese larvae also had Histomonas wenrichi while harboring the heterakids. Two poults (Nos. 2 and 5) had no Heterakis at necropsy, a third had no females, while a fourth poult had only one female which was discarded because it was alone in one cecum. The remaining five poults, all of which had Histomonas wenrichi, had 54 female heterakids, in 47 of which 4201 embryonated eggs were later identified. These 47 worms were fed to nine parasite-free poults, each of which received two to seven such worms, all from the same bird. Subsequently, six of these poults developed infections with H. wenrichi. Therefore, at least 6 of t’he 47 female worms must have had at least one egg that carried the histomonad. Four of the nine recipient poults that received Histomonas wenrichi and Histomonas-free Heterakis larvae, separately, were hosts to worms with such eggs. Two recipient’ poults (Nos. 3 and 4) had at least two such worms. DISCUSSION
AND
CONCLUSIONS
The results of the test that exposed Hetera&s larvae less than 10 days old to Histomonas are not sufficient to warrant a conclusion that such larvae never acquire histomonads and retain them so they are transmitted through the Heterakis eggs. Of the many larvae so exposed (at least 158), only eight were females that survived to maturity. None of these carried eggs that initiated a Histomonas infection in test poults. There are evidences from other sources that acquisition and retention by these young larvae may be quite uncommon. Among those investigators who reported having observed Histomonas in Heterakis, only Kendall (1959) found them in larvae
younger than 10 days. Two reviews of such findings have been published recently (Lee, 1969; Lund, 1969), the latter of which points out that Kendall’s finding represents a different situation, not indicative of recent acquisition. One explanation for the failure of young heterakids to acquire Histomonas wenrichi has been given elsewhere (Lund, 1968). Briefly, this protozoan is almost absent in the tissues, and not very abundant in the cecal lumen until more than 10 clays after its introduction. However, H. meleagridis is often fairly abundant in the lesions of t’he cecal wall 6 or 7 days after infection, and some of the Heterakis larvae are still there. By day 10, almost all of the larvae are in the lumen of the cecum, and H. meleagridis is also there, and sometimes quite numerous, largely because of extrusion from t’he lesions. Nevertheless, there are circumstanccs that tend to make acquisitions less frequent at this early time than later. The smaller, stage 3 larvae probably ingest less material than they will lat’er! and the histomonads are not so abundant in the cecal contents as they will be later. Furt’hermore, t’o be transmit’ted by the Heterakis eggs, the histomonads must persist in t’he worms until the latter mature. Both Siimi (1937) and Lee (1969) appeared to suggest that histomonads that survive long after being ingested have sought out a tissue that is hardly differentiated in Heterakis until Iv-e11 after day 10, the future ovary. Indeed, Siimi believed that histomonads in male worms merely disappear. If they do, presumably it is because they find no tissue appropriate for their preservation. Gibbs (1962) found H. meleagridis in many of the organs of mature male Heterakis. These observations can be harmonized by assuming that as male worms mature, they too differentiate tissues appropriate for the preservation of the histomonads. In contrast to the precarious courses described above for young larvae, the older larvae, juveniles, and young adult hetera-
ACQUISITIOE OF Histomonas kids are frequently afforded fairly good opportunities for acquiring H. meleagridis, and retaining them so that transmission by the embryonated eggs may occur. In this study, four of nine chickens in which Histo,monas-free Heterakis and Histomonas meleagridis were brought together produced worms with eggs that transmitted H. meleagridis. Niimi had only two such instances in 84 chickens. He fed 55 female Heteralcis to seven test chicks to get these two positives. In our test, 37 females with a total of 2366 embryonated eggs were given to nine poults, five of which developed infections with H. meleagridis. Thus, at least five of the 37 females must have had eggs that embryonated and transmitted H. meleagridis that retained viability sufficient to initiate detectable infections. For H. wenrichi the degree of transmission was comparable. Six of nine poults in which Histomonas-free Heterakis and Histomonas wenrichi were associated were later found to have heterakids with eggs that transmitted this protozoan. The six of 47 female worms carrying such eggs represent a ratio almost identical to that for worms carrying H. meleagtidis (five of 37). The results of these studies substantiate the conclusion drawn from the earlier test on acquisition (Lund, 1968) : that single simultaneous infections with Heterakis and Histomonas are entirely adequate to assure the perpetuation of the parasites, but the incidence of infection with Histomonas is low in both the definitive host and the intermediate host. To those interested in the purposeful control of histomoniasis, these circumstances suggest at least the following : 1. The complete exclusion of Histomonas from birds in restricted areas may be attainable by employing biological control through the use of appropriate management. 2. If drug control is to be used primarily or exclusively to prevent or reduce contaimination of soil with HGtomonas-bearing
RY Heterakis
65
eggs of Heterakis, an antihistomonal drug that is effect’ivc only in the lumen of the cecum may be adequate. This could bc a drug of low solubility, so that its presence in the tissues or elimination through the kidneys is inconsequential. Obviously, such a drug would not be selected for use with a species or breed of bird that was so susccptible to histomoniasis as to require that proliferation of the protozoa in the tissues bc halted by use of the drug.
REFERENCES B. J. 1962. The occurrence of the protozoan parasite Histomonns meleclgridis in the adults and eggs of the cecal worm Heternkis gallinae.
GIBBS,
Jourtlal HONGBERG,
of Protozoozogy
9,288-293.
B. M., AXD KULDOVA, J. 1969. Structure of a non-pathogenic histomonad from the cecum of galliform birds and revision of the trichomonad family Monocercomonadidae Kirhg. Journal of Protozoology 16,526535. KIGXDALL, S. B. 1959. The occurrence of Histomo~~us meleagridis in Heternkis gallinae. Pnrnsitology 49,16%172.
1~~.
D. L. 1969. The structure and development of Histomonas melengridis (Mastigamoehidae : Protozoa) in the female reproductive tract of its intermediate host, Heterakis gnllinarum (Kematoda). Pnmsitologv 59, 877-884. J,UND, E. E. 1963. Histomorzas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a nonpathogenic parasite of gallinaceous birds. Jozmnl of Protozoology 10,401-404. I,r:su, E. E. 1967. Response of four breeds of chickens and one breed of turkeys to experimental Heternkti and Histomonns infections. Avtin Diseases 11, 491-502. LUKD. E. E. 1968. Acquisition and liberation of H&omonns wenrichi by Heterakis gnllinnrum. Experimental Parasitology 22, 62-67. I,ITXD. E. E. 1969. Histomoniasis. Advances in Veterinrrry Science and Comparative Medicine 13,35&390. LUND. E. E.. AND BURTNER, R. H., JR. 1957. Infectivity of Hr terakis gallinne eggs with Histomonns mele~rgridis. Experimental Parasitology 6, 189-193. NIIYI. D. 1937. Studies of blackhead. II. Mode of Infection. Journal of the Japanese Society of Veterinargl Science 16, 183-239. TYZZER, E. E., AXD COLLIER, J. 1925. Induced and natural transmission of blackhead in the ahsence of Zieterakis. Journal of Infectious DCeases 31,265-276.