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Transfer of ten-day Heterakis gallinarum larvae: Effect on retention and development of the heterakids, and liberation of Histomonas and Parahistomonas
EXPERIMENTAL
Transfer
31, 361-369 (1972)
PARdSITOLOGY
of Ten-day Heterakis gallinarum Larvae: and Development of the Heterakids, and Histomonas and Parahistomonas Everett
E. Lund and
Anne
Effect on Retention Liberation of
M. Chute
National Animal Parasite Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20706 (Submitted for publication September 16, 1971) LUND, EVERETT AND CHUTE, ANNE M. 1972. Transfer of ten-day Heterakis gallinarum larvae: effect on retention and development of the heterakids, and liberation of Histomonas and Parahistomonas. Experimental Parasitology 31, 361369. Transfer of IOday Heterakis gallinarum larvae from donor chickens to recipient chickens and turkeys resulted in an abrupt loss of approximately half of the transferred worms. However, worms of both sexes surviving to maturity in recipient chickens were larger than in either the recipient turkeys or the control chickens in which the heterakids were allowed to develop undisturbed. Also, female worms recovered from recipient chickens contained substantially more eggs that embryonated than did females recovered from recipient turkeys or control chickens. Histomonas meleagridis was found in the cecal droppings of some recipient birds only 3 days after the larvae were transferred, and the incidence of infection rose rapidly thereafter. The nonpathogenic histomonad, Parahistomonas wenrichi, was also found frequently in recipient birds. A positive correlation was observed between the number of heterakids lost per bird and the incidence of Histomonas infections, suggesting that liberation of histomonads often occurs as the heterakids die. It was also found that far more heterakids carry histomonads t,han liberate them during the normal course of the life cycle. These findings are important in formulating chemical or biological control programs. INDEX
Histomonas meleagridis; Heterakis gallinarum; ParahistoLife Cycle ; Transfer; Transmission; Control ; Chickens ; Turkeys.
DESCRIPTORS:
monas wenrichi;
Histomonas meleagridis and Parahistomonas wenrichi (= H. wenrichi) depend on the common cecal worm, Heterakis gallinarum, for their natural transmission. The latter, in turn, is often transmitted by the ingestion of infected earthworms (Lund et al. 1966). Recent studies (Lund 1968, 1971) have shown that heterakids usually acquire and liberate histomonads during rather short periods in the worms’ life history, when they are 10-20 days old. In certain of these studies the heterakids had started their development in one bird and completed it in another, following transfer by rectal perfusion when the worms were 10 days old. Transfer had been used merely to
fix more precisely the times at which Heterakis acquired and liberated the histomonads. This procedure appeared to affect the survival and development of Heterakis, which in turn seemed to alter the pattern of liberation of Histomonas. The experiments described herein were conducted to study these phenomena. This study required determination of: (1) the immediate loss of heterakids in chickens and turkeys as the result of the transfer procedure, (2) the normal retention and development of worms (not transferred) and the accompanying incidences of infection with Histomonas meleagdis and P. wenrichi, and (3) the retention and de-
361 Copyright
0
1972 by Academic
Press,
Inc.
LUND AND CHUTE
362
velopment of transferred worms and accompanying incidences of H. meleagridis and P. wenrichi. MATERIALS
AND METHODS
Birds. All chickens were New Hampshires and all turkeys were Beltsville Small Whites, from strains reared for many years at the National Animal Parasite Laboratory (formerly the Beltsville Parasitological Laboratory). All birds were incubatorhatched, brooded 4 weeks on wire, and then held in wire-floored cages until use, when they were about 6 weeks old. During the tests, the birds were housed individually in wire-floored cages to permit positive identification of their cecal droppings. Heterakis. The heterakids had been maintained solely in cage-raised chickens during the last two or more passages. With inocula of 90-200 embryonated eggs per bird, the average recovery of lo-day-old larvae had been about 55%. Recovery of mature worms (35-56 days) had been 12-14% when the incidence of Histomonas meleagridis was high (92%) and up to 26% when the incidence of infection with H. meZeagridis was lower (21-440/o). Histomonads were sufficiently numerous in the Heterakis eggs so that a dose of 200 embryonated eggs produced 85-100% incidence of infection with Histomonas meleagridis. Parahistomonas wenrichi was also present, but was less prevalent. To obtain infective material for this study, heterakids were washed from the chickens’ ceca, counted, and sexed. Gravid females were placed in 0.5% formalin in Petri dishes and kept at room temperature for 1421 days to permit embryonation of eggs. Histomonads. The strain of H. meleagridis transmitted by the heterakids had produced clinical histomoniasis but no mortality in 6- to lo-week-old chickens 8-12 days postinoculation. Infections with the nonpathogenic P. wenrichi appeared later (15-23 days postinoculation), as is charac-
teristic for this organism when H. meleagridis is also present. Procedure. Inocula were prepared by rupturing the previously mentioned gravid heterakids in 0.85% saline with a small electric blender, counting embryonated eggs in twenty O.l-ml samples of the resulting suspension, and adjusting the concentration of eggs to the desired number per milliliter. In all instances, the egg suspensions were given by pipette to the crop, after the birds had fed (to insure sufficiently slow passage through the digestive tract so the eggs could hatch). Birds to be donors of larvae were inoculated with Heterakis egg suspensions and necropsied 10 days later. Each was processed individually, with one person examining the cecal contents microscopically for Histomonas and recording host responses thereto; two other workers, using dissecting microscopes, recovered and counted the He terakis larvae and washed them until they were free of all visible cecal material. This required at least six changes of warm 0.85% saline. Birds to receive these larvae were prepared by irrigation of t’he rectum about ‘/ hr before the rectal inoculation (Tyzzer and Collier 1925) with the clean Heterakis larvae. Using this system, the larvae were transferred from donor to recipient bird in only 15-20 min. To insure the flow of material into the ceca (Browne 1922), recipient birds were suspended head downward for y2 hr after inoculation. Control birds were inoculated exactly as were the donor birds. However, the heterakids in control birds were allowed to remain in their original host bird, rather than being transferred at 10 days after inoculation. During the tests, the cecal discharges of the donor, recipient, and control birds were examined daily for Histomonas meleagridis and P. wenrichi. These birds were also observed daily for signs of histomoniasis. At necropsy the control and recipient birds
TRANSFER
OF TEN-DAY
Heterakis
were examined for gross pathological changes in the ceca and liver and for the presence of H. meleagridis and P. wenrichi. Mature heterakids were washed from the ceca, counted, measured, and sexed. The females were processed as described earlier in this section, so that the average number of embryonated eggs per female could be determined. Calculations. Because birds on the major
gallinarum
363
LARVAE
tests were not to be necropsied until their heterakids were mature, a preliminary test was conducted to determine the extent of the Heterakis losses during the first few days after transfer. Thus, the percentage recoveries of Heterakis 2 and 4 days after transfer to chickens or turkeys on the major tests (Figs. 3, 4) were calculated from values obtained on the preliminary test (Fig. 1).
HETERAKIS
ae
LOSS IN DONORS VZHICKENS): A TO 8=97%-79%:19% 19% OF 203~39 LARVAE 203
RECIPIENTS: CHICKENS
TURKEYS
L A:;AE ’
0
-LiYS
:FTER:NOC::ATION
Ld;AE
100 HY
100
90
90
80
80
70 60 50 40 30 20 IO
m 60 50 40 30 20 IO
hi
:~BRd&ATED
::GS-
12
0
2
0
2
DAYS AFTER
4 TRANSFER
14
OF LARVA:
FIG. 1. Results of the preliminary transfer test. Key: shaded area = calculated period when Histomonas was liberated; ~ = avg retention of Heterakis; __ _ = cumulative incidence of Histomonas; 0 = point based on observations ; 0 = point based on calculations; for explanation of Heterakis loss value, see Materials and Methods. HETER..S LOSS: A TO 8=31%=46 LARVAE
0 2 4 6 810
DAYS AFTER
15
20
25
30
35
38
INOCULATION
FIG. 2. Results in control chickens (Heterakis larvae not area = calculated period when Histomonas was liberated; = = cumulative incidence of Hktomonas; ------~ Parahistomonas; l = point based on observations; 0 = point explanation of Heterakis loss value, see Materials and Methods.
transferred). Key: shaded avg retention of Heterakis; = cumulative incidence of based on calculations; for
364
LUND
AiVD
CHUTE
HETERAKIS LOSS DONORS: RECIPIENTS: n TO 8=31%=46 LARVAE a TO 8=X%=39 WORMS I50 TRANSFER: 75 LARVAE
0 2 4 --DAYS
6
8 IO AFTER
15 TRANSFER
20 25 OF LARVAE-
3. Results of transferring lo-day Heterakis larvae from donor to FIG. Key: shaded areas = calculated periods when Histomonas was liberated; --__ = cumulative incidence of Htitomonas; tion of Heterakis; lative incidence of Parahistomonas; 0 = point based on observations; on calculations; for explanation of Heterakis loss value and point at *. Methods and Results, respectively. HETERAKIS DONORS: ATO BB0%=45
150 EMS EGGS
--
‘DAYS
recipient chickens. __ = avg reten----= cumu0 = point based see Materials and
LOSS RECIPIENT%
LARVAE
a TOB-24%=36 WORMS
TRANSFER: 69 LAR”Az
/ i
v
28
/
..............’..........*..* I.’
WITH EMBRYONATED EGGS-AFTER INOCiLATlON 25 20 0 2 4 6 8 IO 12 14 -DAYS AFTER TRANSFER OF LARVAE-
28
FIG. 4. Results of transferring lo-day Heteraki-s larvae from donor chickens to recipient turkeys. Key: shaded areas = calculated periods when Histomonas was liberated; = ----= avg retention of Heterakis; ~-__ = cumulative incidence of Histomonas; cumulative incidence of Parahistomonas; l = point based on observations; 0 = point based on calculations; for explanation of Heterakis loss value, see Materials and Methods.
The periods during which Histomonas meleagridis was liberated were calculated for each test as follows. Hatching of heterakid eggs and migration of larvae to the ceca occupy most of the first 24 hr postinoculation (Dorman 1928; Clapham 1933; Roberts 1937), so liberations of H. meleagridis resulting in the establishment of infections were considered to begin at the
end of the first day. As will be seen, the prepatent period for H. meleagridis can be as short as 3 days. Thus, liberations were considered to have occurred at least 3 days before infections were detected, but not during the first 24 hr postinoculation of Heterakis eggs. These periods of liberation are represented by the shaded areas of Figs. l-4.
TRANSFER OF TEN-DAYHeterakis TABLE
gaharum
LARVAE
365
1
Results and Calculations Indicating a Relationship Between Average Numbers of Histomonas Infections in Birds on all Tests
of Heterakis and
Incidence
Donors Group
‘zi$-
Controls
for for chickens turkeys
Chickens
Chickens
Chickens Claickens
14 203 0 100
25 150 0 96
23 150 0 78
39
46
203 39
No. of birds No. of embryonated eggs given per bird No. of lo-day Heterakis given per bird y. Histomonas infection No. of worms lost per bird as Histomonas infections were initiated No. of larvae given divided by To Histomonas infection No. of larvae lost divided by To Histomonas infection
In each test, the approximate number of heterakids lost per bird during the period of liberation of His tomonas meleagridis was calculated graphically: in each figure, the distance A to B marks the intersections of the line representing the progressive loss of worms with the limits of these periods of liberation. The average number of heterakids lost was then determined from the percentage embraced by the distance A to B (Figs. 14). RESULTS Table 1 shows the number of birds, inoculum, incidence of histomoniasis, and number of heterakids lost during the liberation of H. meleagridis in each test. Table 2 shows the average length of mature heterakids recovered from the control and recipient birds, and the average number of eggs that embryonated per female. Preliminary test to determine losses of heterakids through 4 days after transfer. Because numerous larvae were sought for transfer, 203 Heterakis eggs had been given to each of the 14 donor chickens. All of these birds had infections with Histomonas meleagridis as determined by necropsy 10 days postinoculation (Fig. 1). The period of
Recipients
Chickens
TWkeys
20 150 0 75
20 0 75 95
20 0 69 95
46
45
39
36
156
192
200
79
73
48
59
60
41
38
Development Recovered
of from
TABLE 2 SB-day Heterakis Control
Group :
Avg length, mm Males Females Avg no. eggs that embryonated per female
gallinarum
and Recipient
Controls
Birds
Recipients
9.5 11.1
9.8 11.7
9.1 10.6
34
61
42
liberation of H. meleagridis can be considered to have been Days 2-7, inclusive. The distance A to B embraces a range of 19% of the potential infective dose of 203 embryonated eggs, or 39 larvae. In other words, about 39 larvae were lost by each bird as it acquired its histomonads. It is assumed that most of these lo-day larvae were 4th stage; however, some 3rd stage larvae were present. Collectively, these 14 donor chickens had 1955 heterakids at necropsy, a recovery of 68.8%. Seventy-six larvae were dead or were lost in washing. Ten recipient chickens were given a total of 948 larvae (range: 24-173 each), and 10 recipient turkeys were given
366
LUND
AND
a total of 931 larvae (range: 34-176 each). Five recipient birds of each species were killed during the first 2 days, and the remaining birds were killed 3 and 4 days after having received the larvae. Based on the number of larvae transferred, retention of larvae by chickens was 55.4% during the first 2 days and 48.9% from 3-4 days after transfer. Retention of larvae by recipient turkeys was somewhat better: 65.3% and 62.4%, respectively. Retention is shown on Fig. 1 as a percentage of the larvae transferred. Test to determine the normal retention and development of Heterakis and accompanying incidences of infection with Histomonas meleagridis and P. wenrichi in chickens (control birds). In 25 chickens each given 150 embryonated Heterakis eggs, the worms were permitted to develop to maturity in the original host. Seven days postinoculation, Histomonas meleagridis was detected in the cecal discharges of one bird (Fig. 2). Thereafter the incidence of detectable infections rose rapidly, reaching 96% on the 10th day. During the calculated period of liberation of H. meleagridis, Heterakis losses were calculated to be 31% of 150, or about 46 larvae. (The average retention of Heterakis at 10 days postinoculation was assumed to equal that for the 43 donor chickens given the same inoculum; that average was 48%.) Parahistomonas wenrichi was found in the cecal droppings of one bird at 14 days, and by the 38th day, the incidence of infection had reached 65%. At necropsy, eight of the 25 birds had no Heterakis, but the remainder had a total of 382 worms, a recovery of 10.2% of the 3750 eggs given 38 days earlier. Test to determine the retention and development of Heterakis transferred from donor to recipient chickens, and accompanying incidences of infection with Histomonas meleagridis and P. wenrichi. Of 23 donor chickens each given 150 embryonated Heterakis eggs (inoculum identical to that given the controls), only 18 developed
CHUTE
infections with Histomonas meleagridis detectable at necropsy on the 10th day (Fig. 3). These infections followed liberations that occurred as a calculated worm loss of 31%, or 46 larvae, was taking place. Worm recovery from the 23 chickens averaged 70 (46.7%) but three birds failed to meet a predetermined requirement that each donor have more than 10 larvae. These three, collectively, had but 13 worms. The number of larvae transferred to the 20 recipient chickens averaged 75 (50% of the original 150 given in eggs). H. meleagridis was found in the cecal discharges of two recipient chickens 3 days after transfer of the larvae. The incidence of infection rose rapidly, so that by the 8th day, all except one of the recipient chickens (95%) were voiding histomonads or were clinically ill. These infections followed liberations that occurred during the first 5 days after transfer, as an average of 39 heterakids were lost. Infections with P. wenrichi were first observed on the 5th day after transfer and eventually appeared in all except three (85%) of the recipient chickens. Sixteen birds had infections with both species, but not always simultaneously. All 20 birds were infected with at least one of the two protozoa. Recovery of 38-day Heterakis (i.e., 28 days after t’ransfer) averaged 11.8, or 7.9% of the original inoculum and 15.7% of the larvae transferred. Test to determine the retention and development of Heterakis transferred from donor chickens to recipient turkeys and the accompanying incidences of infection with Histomonas meleagridis and P. wenrichi. Of 20 donor chickens each given 150 embryonated Heterakis eggs (inoculum identical to that used in the two previous tests, only 15 developed infections with Histomonas meleagridis detectable at necropsy on the 10th day (Fig. 4.) The calculated number of larvae lost while these histomonads were being liberated was 45. Worm recovery from the 20 chickens averaged 75.7 (50.5%), and the larvae transferred averaged 69.4 (46.2%) of
TRANSFER OF TEN-DAY Heterakis
the original 150 in eggs. H. meleagridis was found in the cecal discharges of 3 of the 15 recipient turkeys 3 days after transfer of the larvae. By the 7th day after transfer, 75% of the turkeys were voiding histomonads or were clinically ill. The incidence of infection reached 95% on the 14th day, and by the 17th day, four turkeys had died of histomoniasis. The histomonads initiating these infections were liberated as a calculated 36 Heterakis were lost, Parahistomonas wenrichi was first found on the 6th day after transfer, and eventually it appeared in 15 of the 20 recipient turkeys. Recovery of 38-day Heterakis (i.e., 28 days after transfer) averaged 6.9 worms per bird, or 4.6% of the original inoculum and 9.9% of the larvae transferred. DISCUSSION
Preliminary transfer test. The preliminary test established points of reference with regard to the percentage retention of Heterakis 2 and 4 days after transfer to a new host. The calculations of the period of liberation of Histomonas meleagridis and the number of heterakids lost during this period were also introduced. These calculations are useful for comparisons but are not absolute values. Controls. The data obtained from the control group established points of reference with regard to three features: (1) the overall loss of heterakids during the 38-day period in the same host, (2) the pattern of the rise in incidence of H. meleagridis, and (3) comparable information about P. wenrichi. The persistence of infections with H. meleagridis in 24 of the 25 chickens delayed the appearance of P. wenrichi in most birds, the latter organism being known not to thrive while H. meleagridis is quite active (Lund 1963). Transfer of lo-day Heterakis from donor to recipient chickens. The early and precipitous rise in detections of Histomonas meleagridis following the heterakid transfer can only indicate liberations of the proto-
gallinarum
LARVAE
367
zoa at least 3 days earlier. Indeed, 70% of the birds acquired their histomonads from liberations occurring within the first 2 days after the transfer, the time during which most worms were lost. As worm losses diminished, so did the liberations of H. meleagridis, as adjudged from the appearance of new infections. The incidence of P. wenrichi, which also rose sharply (starting the 5th day after transfer of the larvae), was almost certainly influenced by the cecal lesions that developed in so many of the recipient chickens as infections with H. meleagridis passed through the acute stage (Lund 1967). The ultimate rise of the incidence of P. wenrichi to 85% indicates that this organism, like H. meleagridis, is liberated with considerable frequency following transfer of lo-day Heterakis larvae. Transfer of lo-day Heterakis from donor chickens to recipient turkeys. The similarities in Figs. 3 and 4 are evident; however, there are also differences. Worm recovery from the donors providing larvae for recipient turkeys was slightly better than in donors providing larvae for recipient chickens. Fifty percent of the turkeys acquired their histomonads from liberations occurring within the first 2 days, the time during which the rate of Heterakis loss was greatest. As the rate of this loss decreased, the rise in Histomonas infection again became more gradual. The incidence of H. meleagridis culminated at 95%, as was the case with the recipient chickens, but it took more than twice as long (14 days after transfer of larvae) to attain this incidence in the turkeys. During the 11 days in which histomonads initiating these infections were liberated, a calculated average of only about 36 heterakids per bird were lost. These losseswere probably due to the hardships imposed by the transfer procedures. Later losses,when all except one turkey had active infections with Histomonas and some were severely ill, must have occurred primarily because the ceca no longer afforded
368
LUND AND CHUTE
a tenable habitat for the worms. The influence of cecal involvement on the survival of Heteralcis is almost always greater in young turkeys than in young chickens (Lund 1958; Lund and Chute 1970). In this study also, the loss rate in turkeys after the 4th day following transfer was greater than the loss rate in chickens for the same period of time (Fig. 4 compared with Fig. 3). Because the number of recipient birds of each species that could be necropsied for worm recovery 2,4 and 28 days after transfer was relatively small, differences are not statistically significant. The incidence of the nonpathogenic histomonad, Parahistomonas wenrichi, rose sharply at first (6 and 7 days after the heterakid transfer). Then, almost certainly because of the effects of the Histomonas infect.ions, the rise in incidence slowed and consistently remained lower than that in the recipient chickens. Influence of transfer on development of Heterakis and liberation of histomonads. Whatever adversities Heterakis encounters, by transfer or otherwise, one must assume that if some individuals survive while others succumb to the adversity, it is largely the fittest, the most hardy, that survive. The average lengths of heterakids from recipient chickens were slightly greater than the averages for undisturbed worms of the corresponding sex taken from the chickens of the control group, all collected at 38 days (Table 2). This is not because maximum lengths were greater in recipient birds, but rather because of the elimination of disproportionate numbers of worms that would have lowered the average, had they remained. Throughout, we have been calling attention to the number of Heterakis lost as infections with Histomonas were initiated. If any relationship exists between the numbers of Heterakis larvae entering a bird and that bird’s chances of developing an infection with Histomonas, the protozoan must escape from worms that live, from worms
that, die, or from both. The issue is important because some measures for the control of Histomonas infections destroy the heterakids while they are still in the bird. We have only one opportunity to determine the number of worms living in a bird: at necropsy. If this occurs late, and Histomonas has been liberated and multiplied sufficiently to provoke a severe cecal response, heterakids are lost as a result of the bird’s ceca becoming an untenable habitat for the worms. Consequently, we have had to relate the incidence of Histomonas infections with (1) the number of larvae or embryonated Heterakis eggs given to the birds (a relationship used for many years) ; and (2) the number of heterakids apparently lost during the period when histomonads that initiated infections were being liberated (a newly introduced relationship). The Iast two lines in Table 1, calculated from t’he other values shown for each group, represent these relationships. Apparently, the results for the six groups are more consistent with respect to the relationship between incidence of Histomonas infections and numbers of heterakids lost (final line in Table 1). However, one need not assume that infections arise only from worms that perish. Tentatively, we believe that the data suggest that effective liberations are predominantly from such worms. Unlike Parahistomonas wenrichi, which appears to be unable to invade tissues or muhiply therein and may also be unable to escape from livcan cering tissue, Histomonas meleagridis tainly invade living tissue and may also be able to escape from it (the Heterakis larvae, for example). Perhaps the most important information to emerge from this study is the disclosure that far more heterakids are carrying histomonads than liberate them during the normal course of the life cycle. It is quite possible that even those worms that survive the transfer may still be carrying histomonads that could be liberated under appropriate circumstances. In any control program,
TRANSFER OF TEN-DAY Heterakis
chemical or biological, such contingencies are important. ACKNOWLEDGMENT The authors are grateful to Mr. Gary C. Wilkins for technical assistance. REFERENCES BROWNE, T. G. 1922. Some observations
on the digestive system of the fowl. Journal of Comparative Pathology and Therapeutics 35, 1232. CLAPHAM, P. A. 1933. On the life-history of Heterakis gallinae. Journal of Helminthology 11, 6786. DORMAN, H. P. 1928. Studies on the life cycle of Heterakis papillosa (Bloch). Transactions of the American Microscopical Society 47, 379413. LUND, E. E. 1958. Growth and development of Heterakis gallinae in turkeys and chickens infected with Histomonas meleagridis. Journal of Parasitology 44,297-301. LUND, E. E. 1963. Histomonas
wenrichi n. sp. (Mastigophora: Mastigamoebidae), a nonpathogenic parasite of gallinaceous birds. Journal of Protozoology 10,401404. LUND, E. E. 1967. Response of four breeds of chick-
gcdlinarzm
LARVAE
369
ens and one breed of turkeys to experimental Heterakis and Histomonas infections. Avian Diseases 11,491~502. LUND, E. E. 1968. Acquisition and liberation of Histomonas wenrichi by Heterakis gallinarum. Experimental Parasitology 22,62-67. LUND, E. E. 1971. Histomonas meleagridis and H. wenrichi: Time of acquisition by Heterakis gallinarum. Experimental Parasitology 29,5965. LUND, E. E., AND CHUTE, A. M. 1970. Relative importance of young and mature chickens and turkeys in contaminating soil with Histomonas-bearing Heterakis eggs. Avian Diseases
14,342-348. LUND, E. E., WEHR, E. E. AND ELLIS, D. J. 1966. Earthworm transmission of Heterakis and
Histomonas to turkeys and chickens. Journal of Parasitology 52,899-902. ROBERTS, F. H. S. 1937. Studies on the life history and economic importance of Heterakis gallinae
(Gmelin, 1790, Freeborn, 1923), the caecum worm of fowls. Australian Journal of Experimental Biology and Medical Science 15, 429439. TYZZER, E. E., AND COLLIER, J. 1925.Induced and natural transmission of blackhead in the absence of Heterakis. Journal of Infectious Diseases 37,265-276.
Transfer
31, 361-369 (1972)
PARdSITOLOGY
of Ten-day Heterakis gallinarum Larvae: and Development of the Heterakids, and Histomonas and Parahistomonas Everett
E. Lund and
Anne
Effect on Retention Liberation of
M. Chute
National Animal Parasite Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20706 (Submitted for publication September 16, 1971) LUND, EVERETT AND CHUTE, ANNE M. 1972. Transfer of ten-day Heterakis gallinarum larvae: effect on retention and development of the heterakids, and liberation of Histomonas and Parahistomonas. Experimental Parasitology 31, 361369. Transfer of IOday Heterakis gallinarum larvae from donor chickens to recipient chickens and turkeys resulted in an abrupt loss of approximately half of the transferred worms. However, worms of both sexes surviving to maturity in recipient chickens were larger than in either the recipient turkeys or the control chickens in which the heterakids were allowed to develop undisturbed. Also, female worms recovered from recipient chickens contained substantially more eggs that embryonated than did females recovered from recipient turkeys or control chickens. Histomonas meleagridis was found in the cecal droppings of some recipient birds only 3 days after the larvae were transferred, and the incidence of infection rose rapidly thereafter. The nonpathogenic histomonad, Parahistomonas wenrichi, was also found frequently in recipient birds. A positive correlation was observed between the number of heterakids lost per bird and the incidence of Histomonas infections, suggesting that liberation of histomonads often occurs as the heterakids die. It was also found that far more heterakids carry histomonads t,han liberate them during the normal course of the life cycle. These findings are important in formulating chemical or biological control programs. INDEX
Histomonas meleagridis; Heterakis gallinarum; ParahistoLife Cycle ; Transfer; Transmission; Control ; Chickens ; Turkeys.
DESCRIPTORS:
monas wenrichi;
Histomonas meleagridis and Parahistomonas wenrichi (= H. wenrichi) depend on the common cecal worm, Heterakis gallinarum, for their natural transmission. The latter, in turn, is often transmitted by the ingestion of infected earthworms (Lund et al. 1966). Recent studies (Lund 1968, 1971) have shown that heterakids usually acquire and liberate histomonads during rather short periods in the worms’ life history, when they are 10-20 days old. In certain of these studies the heterakids had started their development in one bird and completed it in another, following transfer by rectal perfusion when the worms were 10 days old. Transfer had been used merely to
fix more precisely the times at which Heterakis acquired and liberated the histomonads. This procedure appeared to affect the survival and development of Heterakis, which in turn seemed to alter the pattern of liberation of Histomonas. The experiments described herein were conducted to study these phenomena. This study required determination of: (1) the immediate loss of heterakids in chickens and turkeys as the result of the transfer procedure, (2) the normal retention and development of worms (not transferred) and the accompanying incidences of infection with Histomonas meleagdis and P. wenrichi, and (3) the retention and de-
361 Copyright
0
1972 by Academic
Press,
Inc.
LUND AND CHUTE
362
velopment of transferred worms and accompanying incidences of H. meleagridis and P. wenrichi. MATERIALS
AND METHODS
Birds. All chickens were New Hampshires and all turkeys were Beltsville Small Whites, from strains reared for many years at the National Animal Parasite Laboratory (formerly the Beltsville Parasitological Laboratory). All birds were incubatorhatched, brooded 4 weeks on wire, and then held in wire-floored cages until use, when they were about 6 weeks old. During the tests, the birds were housed individually in wire-floored cages to permit positive identification of their cecal droppings. Heterakis. The heterakids had been maintained solely in cage-raised chickens during the last two or more passages. With inocula of 90-200 embryonated eggs per bird, the average recovery of lo-day-old larvae had been about 55%. Recovery of mature worms (35-56 days) had been 12-14% when the incidence of Histomonas meleagridis was high (92%) and up to 26% when the incidence of infection with H. meZeagridis was lower (21-440/o). Histomonads were sufficiently numerous in the Heterakis eggs so that a dose of 200 embryonated eggs produced 85-100% incidence of infection with Histomonas meleagridis. Parahistomonas wenrichi was also present, but was less prevalent. To obtain infective material for this study, heterakids were washed from the chickens’ ceca, counted, and sexed. Gravid females were placed in 0.5% formalin in Petri dishes and kept at room temperature for 1421 days to permit embryonation of eggs. Histomonads. The strain of H. meleagridis transmitted by the heterakids had produced clinical histomoniasis but no mortality in 6- to lo-week-old chickens 8-12 days postinoculation. Infections with the nonpathogenic P. wenrichi appeared later (15-23 days postinoculation), as is charac-
teristic for this organism when H. meleagridis is also present. Procedure. Inocula were prepared by rupturing the previously mentioned gravid heterakids in 0.85% saline with a small electric blender, counting embryonated eggs in twenty O.l-ml samples of the resulting suspension, and adjusting the concentration of eggs to the desired number per milliliter. In all instances, the egg suspensions were given by pipette to the crop, after the birds had fed (to insure sufficiently slow passage through the digestive tract so the eggs could hatch). Birds to be donors of larvae were inoculated with Heterakis egg suspensions and necropsied 10 days later. Each was processed individually, with one person examining the cecal contents microscopically for Histomonas and recording host responses thereto; two other workers, using dissecting microscopes, recovered and counted the He terakis larvae and washed them until they were free of all visible cecal material. This required at least six changes of warm 0.85% saline. Birds to receive these larvae were prepared by irrigation of t’he rectum about ‘/ hr before the rectal inoculation (Tyzzer and Collier 1925) with the clean Heterakis larvae. Using this system, the larvae were transferred from donor to recipient bird in only 15-20 min. To insure the flow of material into the ceca (Browne 1922), recipient birds were suspended head downward for y2 hr after inoculation. Control birds were inoculated exactly as were the donor birds. However, the heterakids in control birds were allowed to remain in their original host bird, rather than being transferred at 10 days after inoculation. During the tests, the cecal discharges of the donor, recipient, and control birds were examined daily for Histomonas meleagridis and P. wenrichi. These birds were also observed daily for signs of histomoniasis. At necropsy the control and recipient birds
TRANSFER
OF TEN-DAY
Heterakis
were examined for gross pathological changes in the ceca and liver and for the presence of H. meleagridis and P. wenrichi. Mature heterakids were washed from the ceca, counted, measured, and sexed. The females were processed as described earlier in this section, so that the average number of embryonated eggs per female could be determined. Calculations. Because birds on the major
gallinarum
363
LARVAE
tests were not to be necropsied until their heterakids were mature, a preliminary test was conducted to determine the extent of the Heterakis losses during the first few days after transfer. Thus, the percentage recoveries of Heterakis 2 and 4 days after transfer to chickens or turkeys on the major tests (Figs. 3, 4) were calculated from values obtained on the preliminary test (Fig. 1).
HETERAKIS
ae
LOSS IN DONORS VZHICKENS): A TO 8=97%-79%:19% 19% OF 203~39 LARVAE 203
RECIPIENTS: CHICKENS
TURKEYS
L A:;AE ’
0
-LiYS
:FTER:NOC::ATION
Ld;AE
100 HY
100
90
90
80
80
70 60 50 40 30 20 IO
m 60 50 40 30 20 IO
hi
:~BRd&ATED
::GS-
12
0
2
0
2
DAYS AFTER
4 TRANSFER
14
OF LARVA:
FIG. 1. Results of the preliminary transfer test. Key: shaded area = calculated period when Histomonas was liberated; ~ = avg retention of Heterakis; __ _ = cumulative incidence of Histomonas; 0 = point based on observations ; 0 = point based on calculations; for explanation of Heterakis loss value, see Materials and Methods. HETER..S LOSS: A TO 8=31%=46 LARVAE
0 2 4 6 810
DAYS AFTER
15
20
25
30
35
38
INOCULATION
FIG. 2. Results in control chickens (Heterakis larvae not area = calculated period when Histomonas was liberated; = = cumulative incidence of Hktomonas; ------~ Parahistomonas; l = point based on observations; 0 = point explanation of Heterakis loss value, see Materials and Methods.
transferred). Key: shaded avg retention of Heterakis; = cumulative incidence of based on calculations; for
364
LUND
AiVD
CHUTE
HETERAKIS LOSS DONORS: RECIPIENTS: n TO 8=31%=46 LARVAE a TO 8=X%=39 WORMS I50 TRANSFER: 75 LARVAE
0 2 4 --DAYS
6
8 IO AFTER
15 TRANSFER
20 25 OF LARVAE-
3. Results of transferring lo-day Heterakis larvae from donor to FIG. Key: shaded areas = calculated periods when Histomonas was liberated; --__ = cumulative incidence of Htitomonas; tion of Heterakis; lative incidence of Parahistomonas; 0 = point based on observations; on calculations; for explanation of Heterakis loss value and point at *. Methods and Results, respectively. HETERAKIS DONORS: ATO BB0%=45
150 EMS EGGS
--
‘DAYS
recipient chickens. __ = avg reten----= cumu0 = point based see Materials and
LOSS RECIPIENT%
LARVAE
a TOB-24%=36 WORMS
TRANSFER: 69 LAR”Az
/ i
v
28
/
..............’..........*..* I.’
WITH EMBRYONATED EGGS-AFTER INOCiLATlON 25 20 0 2 4 6 8 IO 12 14 -DAYS AFTER TRANSFER OF LARVAE-
28
FIG. 4. Results of transferring lo-day Heteraki-s larvae from donor chickens to recipient turkeys. Key: shaded areas = calculated periods when Histomonas was liberated; = ----= avg retention of Heterakis; ~-__ = cumulative incidence of Histomonas; cumulative incidence of Parahistomonas; l = point based on observations; 0 = point based on calculations; for explanation of Heterakis loss value, see Materials and Methods.
The periods during which Histomonas meleagridis was liberated were calculated for each test as follows. Hatching of heterakid eggs and migration of larvae to the ceca occupy most of the first 24 hr postinoculation (Dorman 1928; Clapham 1933; Roberts 1937), so liberations of H. meleagridis resulting in the establishment of infections were considered to begin at the
end of the first day. As will be seen, the prepatent period for H. meleagridis can be as short as 3 days. Thus, liberations were considered to have occurred at least 3 days before infections were detected, but not during the first 24 hr postinoculation of Heterakis eggs. These periods of liberation are represented by the shaded areas of Figs. l-4.
TRANSFER OF TEN-DAYHeterakis TABLE
gaharum
LARVAE
365
1
Results and Calculations Indicating a Relationship Between Average Numbers of Histomonas Infections in Birds on all Tests
of Heterakis and
Incidence
Donors Group
‘zi$-
Controls
for for chickens turkeys
Chickens
Chickens
Chickens Claickens
14 203 0 100
25 150 0 96
23 150 0 78
39
46
203 39
No. of birds No. of embryonated eggs given per bird No. of lo-day Heterakis given per bird y. Histomonas infection No. of worms lost per bird as Histomonas infections were initiated No. of larvae given divided by To Histomonas infection No. of larvae lost divided by To Histomonas infection
In each test, the approximate number of heterakids lost per bird during the period of liberation of His tomonas meleagridis was calculated graphically: in each figure, the distance A to B marks the intersections of the line representing the progressive loss of worms with the limits of these periods of liberation. The average number of heterakids lost was then determined from the percentage embraced by the distance A to B (Figs. 14). RESULTS Table 1 shows the number of birds, inoculum, incidence of histomoniasis, and number of heterakids lost during the liberation of H. meleagridis in each test. Table 2 shows the average length of mature heterakids recovered from the control and recipient birds, and the average number of eggs that embryonated per female. Preliminary test to determine losses of heterakids through 4 days after transfer. Because numerous larvae were sought for transfer, 203 Heterakis eggs had been given to each of the 14 donor chickens. All of these birds had infections with Histomonas meleagridis as determined by necropsy 10 days postinoculation (Fig. 1). The period of
Recipients
Chickens
TWkeys
20 150 0 75
20 0 75 95
20 0 69 95
46
45
39
36
156
192
200
79
73
48
59
60
41
38
Development Recovered
of from
TABLE 2 SB-day Heterakis Control
Group :
Avg length, mm Males Females Avg no. eggs that embryonated per female
gallinarum
and Recipient
Controls
Birds
Recipients
9.5 11.1
9.8 11.7
9.1 10.6
34
61
42
liberation of H. meleagridis can be considered to have been Days 2-7, inclusive. The distance A to B embraces a range of 19% of the potential infective dose of 203 embryonated eggs, or 39 larvae. In other words, about 39 larvae were lost by each bird as it acquired its histomonads. It is assumed that most of these lo-day larvae were 4th stage; however, some 3rd stage larvae were present. Collectively, these 14 donor chickens had 1955 heterakids at necropsy, a recovery of 68.8%. Seventy-six larvae were dead or were lost in washing. Ten recipient chickens were given a total of 948 larvae (range: 24-173 each), and 10 recipient turkeys were given
366
LUND
AND
a total of 931 larvae (range: 34-176 each). Five recipient birds of each species were killed during the first 2 days, and the remaining birds were killed 3 and 4 days after having received the larvae. Based on the number of larvae transferred, retention of larvae by chickens was 55.4% during the first 2 days and 48.9% from 3-4 days after transfer. Retention of larvae by recipient turkeys was somewhat better: 65.3% and 62.4%, respectively. Retention is shown on Fig. 1 as a percentage of the larvae transferred. Test to determine the normal retention and development of Heterakis and accompanying incidences of infection with Histomonas meleagridis and P. wenrichi in chickens (control birds). In 25 chickens each given 150 embryonated Heterakis eggs, the worms were permitted to develop to maturity in the original host. Seven days postinoculation, Histomonas meleagridis was detected in the cecal discharges of one bird (Fig. 2). Thereafter the incidence of detectable infections rose rapidly, reaching 96% on the 10th day. During the calculated period of liberation of H. meleagridis, Heterakis losses were calculated to be 31% of 150, or about 46 larvae. (The average retention of Heterakis at 10 days postinoculation was assumed to equal that for the 43 donor chickens given the same inoculum; that average was 48%.) Parahistomonas wenrichi was found in the cecal droppings of one bird at 14 days, and by the 38th day, the incidence of infection had reached 65%. At necropsy, eight of the 25 birds had no Heterakis, but the remainder had a total of 382 worms, a recovery of 10.2% of the 3750 eggs given 38 days earlier. Test to determine the retention and development of Heterakis transferred from donor to recipient chickens, and accompanying incidences of infection with Histomonas meleagridis and P. wenrichi. Of 23 donor chickens each given 150 embryonated Heterakis eggs (inoculum identical to that given the controls), only 18 developed
CHUTE
infections with Histomonas meleagridis detectable at necropsy on the 10th day (Fig. 3). These infections followed liberations that occurred as a calculated worm loss of 31%, or 46 larvae, was taking place. Worm recovery from the 23 chickens averaged 70 (46.7%) but three birds failed to meet a predetermined requirement that each donor have more than 10 larvae. These three, collectively, had but 13 worms. The number of larvae transferred to the 20 recipient chickens averaged 75 (50% of the original 150 given in eggs). H. meleagridis was found in the cecal discharges of two recipient chickens 3 days after transfer of the larvae. The incidence of infection rose rapidly, so that by the 8th day, all except one of the recipient chickens (95%) were voiding histomonads or were clinically ill. These infections followed liberations that occurred during the first 5 days after transfer, as an average of 39 heterakids were lost. Infections with P. wenrichi were first observed on the 5th day after transfer and eventually appeared in all except three (85%) of the recipient chickens. Sixteen birds had infections with both species, but not always simultaneously. All 20 birds were infected with at least one of the two protozoa. Recovery of 38-day Heterakis (i.e., 28 days after t’ransfer) averaged 11.8, or 7.9% of the original inoculum and 15.7% of the larvae transferred. Test to determine the retention and development of Heterakis transferred from donor chickens to recipient turkeys and the accompanying incidences of infection with Histomonas meleagridis and P. wenrichi. Of 20 donor chickens each given 150 embryonated Heterakis eggs (inoculum identical to that used in the two previous tests, only 15 developed infections with Histomonas meleagridis detectable at necropsy on the 10th day (Fig. 4.) The calculated number of larvae lost while these histomonads were being liberated was 45. Worm recovery from the 20 chickens averaged 75.7 (50.5%), and the larvae transferred averaged 69.4 (46.2%) of
TRANSFER OF TEN-DAY Heterakis
the original 150 in eggs. H. meleagridis was found in the cecal discharges of 3 of the 15 recipient turkeys 3 days after transfer of the larvae. By the 7th day after transfer, 75% of the turkeys were voiding histomonads or were clinically ill. The incidence of infection reached 95% on the 14th day, and by the 17th day, four turkeys had died of histomoniasis. The histomonads initiating these infections were liberated as a calculated 36 Heterakis were lost, Parahistomonas wenrichi was first found on the 6th day after transfer, and eventually it appeared in 15 of the 20 recipient turkeys. Recovery of 38-day Heterakis (i.e., 28 days after transfer) averaged 6.9 worms per bird, or 4.6% of the original inoculum and 9.9% of the larvae transferred. DISCUSSION
Preliminary transfer test. The preliminary test established points of reference with regard to the percentage retention of Heterakis 2 and 4 days after transfer to a new host. The calculations of the period of liberation of Histomonas meleagridis and the number of heterakids lost during this period were also introduced. These calculations are useful for comparisons but are not absolute values. Controls. The data obtained from the control group established points of reference with regard to three features: (1) the overall loss of heterakids during the 38-day period in the same host, (2) the pattern of the rise in incidence of H. meleagridis, and (3) comparable information about P. wenrichi. The persistence of infections with H. meleagridis in 24 of the 25 chickens delayed the appearance of P. wenrichi in most birds, the latter organism being known not to thrive while H. meleagridis is quite active (Lund 1963). Transfer of lo-day Heterakis from donor to recipient chickens. The early and precipitous rise in detections of Histomonas meleagridis following the heterakid transfer can only indicate liberations of the proto-
gallinarum
LARVAE
367
zoa at least 3 days earlier. Indeed, 70% of the birds acquired their histomonads from liberations occurring within the first 2 days after the transfer, the time during which most worms were lost. As worm losses diminished, so did the liberations of H. meleagridis, as adjudged from the appearance of new infections. The incidence of P. wenrichi, which also rose sharply (starting the 5th day after transfer of the larvae), was almost certainly influenced by the cecal lesions that developed in so many of the recipient chickens as infections with H. meleagridis passed through the acute stage (Lund 1967). The ultimate rise of the incidence of P. wenrichi to 85% indicates that this organism, like H. meleagridis, is liberated with considerable frequency following transfer of lo-day Heterakis larvae. Transfer of lo-day Heterakis from donor chickens to recipient turkeys. The similarities in Figs. 3 and 4 are evident; however, there are also differences. Worm recovery from the donors providing larvae for recipient turkeys was slightly better than in donors providing larvae for recipient chickens. Fifty percent of the turkeys acquired their histomonads from liberations occurring within the first 2 days, the time during which the rate of Heterakis loss was greatest. As the rate of this loss decreased, the rise in Histomonas infection again became more gradual. The incidence of H. meleagridis culminated at 95%, as was the case with the recipient chickens, but it took more than twice as long (14 days after transfer of larvae) to attain this incidence in the turkeys. During the 11 days in which histomonads initiating these infections were liberated, a calculated average of only about 36 heterakids per bird were lost. These losseswere probably due to the hardships imposed by the transfer procedures. Later losses,when all except one turkey had active infections with Histomonas and some were severely ill, must have occurred primarily because the ceca no longer afforded
368
LUND AND CHUTE
a tenable habitat for the worms. The influence of cecal involvement on the survival of Heteralcis is almost always greater in young turkeys than in young chickens (Lund 1958; Lund and Chute 1970). In this study also, the loss rate in turkeys after the 4th day following transfer was greater than the loss rate in chickens for the same period of time (Fig. 4 compared with Fig. 3). Because the number of recipient birds of each species that could be necropsied for worm recovery 2,4 and 28 days after transfer was relatively small, differences are not statistically significant. The incidence of the nonpathogenic histomonad, Parahistomonas wenrichi, rose sharply at first (6 and 7 days after the heterakid transfer). Then, almost certainly because of the effects of the Histomonas infect.ions, the rise in incidence slowed and consistently remained lower than that in the recipient chickens. Influence of transfer on development of Heterakis and liberation of histomonads. Whatever adversities Heterakis encounters, by transfer or otherwise, one must assume that if some individuals survive while others succumb to the adversity, it is largely the fittest, the most hardy, that survive. The average lengths of heterakids from recipient chickens were slightly greater than the averages for undisturbed worms of the corresponding sex taken from the chickens of the control group, all collected at 38 days (Table 2). This is not because maximum lengths were greater in recipient birds, but rather because of the elimination of disproportionate numbers of worms that would have lowered the average, had they remained. Throughout, we have been calling attention to the number of Heterakis lost as infections with Histomonas were initiated. If any relationship exists between the numbers of Heterakis larvae entering a bird and that bird’s chances of developing an infection with Histomonas, the protozoan must escape from worms that live, from worms
that, die, or from both. The issue is important because some measures for the control of Histomonas infections destroy the heterakids while they are still in the bird. We have only one opportunity to determine the number of worms living in a bird: at necropsy. If this occurs late, and Histomonas has been liberated and multiplied sufficiently to provoke a severe cecal response, heterakids are lost as a result of the bird’s ceca becoming an untenable habitat for the worms. Consequently, we have had to relate the incidence of Histomonas infections with (1) the number of larvae or embryonated Heterakis eggs given to the birds (a relationship used for many years) ; and (2) the number of heterakids apparently lost during the period when histomonads that initiated infections were being liberated (a newly introduced relationship). The Iast two lines in Table 1, calculated from t’he other values shown for each group, represent these relationships. Apparently, the results for the six groups are more consistent with respect to the relationship between incidence of Histomonas infections and numbers of heterakids lost (final line in Table 1). However, one need not assume that infections arise only from worms that perish. Tentatively, we believe that the data suggest that effective liberations are predominantly from such worms. Unlike Parahistomonas wenrichi, which appears to be unable to invade tissues or muhiply therein and may also be unable to escape from livcan cering tissue, Histomonas meleagridis tainly invade living tissue and may also be able to escape from it (the Heterakis larvae, for example). Perhaps the most important information to emerge from this study is the disclosure that far more heterakids are carrying histomonads than liberate them during the normal course of the life cycle. It is quite possible that even those worms that survive the transfer may still be carrying histomonads that could be liberated under appropriate circumstances. In any control program,
TRANSFER OF TEN-DAY Heterakis
chemical or biological, such contingencies are important. ACKNOWLEDGMENT The authors are grateful to Mr. Gary C. Wilkins for technical assistance. REFERENCES BROWNE, T. G. 1922. Some observations
on the digestive system of the fowl. Journal of Comparative Pathology and Therapeutics 35, 1232. CLAPHAM, P. A. 1933. On the life-history of Heterakis gallinae. Journal of Helminthology 11, 6786. DORMAN, H. P. 1928. Studies on the life cycle of Heterakis papillosa (Bloch). Transactions of the American Microscopical Society 47, 379413. LUND, E. E. 1958. Growth and development of Heterakis gallinae in turkeys and chickens infected with Histomonas meleagridis. Journal of Parasitology 44,297-301. LUND, E. E. 1963. Histomonas
wenrichi n. sp. (Mastigophora: Mastigamoebidae), a nonpathogenic parasite of gallinaceous birds. Journal of Protozoology 10,401404. LUND, E. E. 1967. Response of four breeds of chick-
gcdlinarzm
LARVAE
369
ens and one breed of turkeys to experimental Heterakis and Histomonas infections. Avian Diseases 11,491~502. LUND, E. E. 1968. Acquisition and liberation of Histomonas wenrichi by Heterakis gallinarum. Experimental Parasitology 22,62-67. LUND, E. E. 1971. Histomonas meleagridis and H. wenrichi: Time of acquisition by Heterakis gallinarum. Experimental Parasitology 29,5965. LUND, E. E., AND CHUTE, A. M. 1970. Relative importance of young and mature chickens and turkeys in contaminating soil with Histomonas-bearing Heterakis eggs. Avian Diseases
14,342-348. LUND, E. E., WEHR, E. E. AND ELLIS, D. J. 1966. Earthworm transmission of Heterakis and
Histomonas to turkeys and chickens. Journal of Parasitology 52,899-902. ROBERTS, F. H. S. 1937. Studies on the life history and economic importance of Heterakis gallinae
(Gmelin, 1790, Freeborn, 1923), the caecum worm of fowls. Australian Journal of Experimental Biology and Medical Science 15, 429439. TYZZER, E. E., AND COLLIER, J. 1925.Induced and natural transmission of blackhead in the absence of Heterakis. Journal of Infectious Diseases 37,265-276.