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Infectivity of Heterakis gallinae eggs with Histomonas meleagridis
PARASITOLOGY 6, 189-193 (1957)
EXPERIMENTAL
Infectivity with Everett Animal
Disease United
of Heterakis gallinue His tomonas meleagridis E. Lund
and Parasite
and
Roy
H. Burtner,
Research Branch, Agricultural of Agriculture, Beltsville,
States Department
(Submitted for publication,
Eggs Jr. Research Maryland
Service,
3 August 1956)
Histomonas meleagridis, the causative agent of blackhead in poultry, is easily maintained in eggs of the cecal worm, Heterakis gallinae, and blackhead disease is readily produced by the administration of the infected eggs of this nematode. Histomonads have not been discovered inside the cecal worm eggs, however, although a number of investigators have searched for them. Consequently, when an infection of blackhead is experimentally induced by feeding eggs of the cecal worm, the investigator knows only how many embryonated eggs he administers-not how many of them contain histomonads and therefore not how many Protozoa are introduced thereby. The investigation herein reported was conducted to determine what proportion of the eggs produced by cecal worms that develop in the alimentary tract of histomonad-infected birds may be expected to contain these protozoa. The method by which the determinations were made and the findings are summarized below. EXPERIMENTAL
PROCEDURE
Eggs of Heterakis gallinae which had been recovered from birds infected with a non-pathogenic strain of Histomonas were administered orally to three 5-week-old chickens. Histomonads were present in the cecal droppings of two of the inoculated birds from the 15th to the 39th day following the administration of eggs. Both birds were killed on the 41st day after infection. A total of 783 cecal worms, of which nearly 400 were gravid females, was recovered. The females were washed in several changes of 0.85 % salt solution and left intact in saline at room temperature for 16 days to permit the contained eggs to embryonate. At the end of that time about 50 % contained 189
190
EVERETT
E. LUND
AND
ROY H. BURTNER,
JR.
larvae, most of the remainder having failed to develop beyond the early stages of segmentation. New Hampshire Red chickens, 6-weeks-old, were divided into two lots of 60 each. The birds of one lot received whole worms. Those of the other received embryonated eggs taken from a large number of worms. All worms had been treated as described above. Forty-six birds of Lot I received one worm each. The smallest number of embryonated eggs in any worm administered to these birds was 100, the largest was 244, and the average was 160. Thirteen birds received two worms each. The worms selected for administration averaged 112 embryonated eggs each. Each bird received therefore approximately 224 such eggs. The remaining bird of Lot I received three worms, which contained a total of 179 embryonated eggs. In all cases the worms were introduced into the crop along with 1 ml of saline solution. Each of the 60 birds in Lot II received approximately 160 embryonated cecal worm eggs suspended in 1 ml of saline. These eggs were collected from about 100 worms selected from the same source as those given to the birds in Lot I. They were thoroughly mixed and suspended in saline, and the suspension was then calibrated by counting the number of embryonated eggs in 20 low-power fields of the microscope in each of eight 0.02-ml samples of the agitated suspension. Each sample filled the space under a cover glass 22-mm square. The average of the eight counts was then multiplied by a factor calculated to convert the figure to number of eggs per milliliter of suspension. The suspension was then diluted so that 1 ml contained approximately 160 embryonated eggs. Birds from each lot were killed at predesignated intervals from the 8th to the 56th day after infection and examined for both Histomcmas and Heterakis. OBSERVATIONS
AND
INTERPRETATIONS
The more important data are presented in the accompanying table. Because of the small number of birds that acquired infections of Histomonas, the examinations made during the first 2 weeks following experimental infection were not considered conclusive. For this reason, in the table the findings for this period are shown separately from the others. From the 18th day onward, 77 % of the chickens in Lot II were positive for Histomonas at autopsy. This is considered to represent one infection for each 208 embryonated eggs administered. For the corresponding period only 38.4 % of the chickens in Lot I that
HISTOMONAD
INFECTIVITY
OF HETERAKIS
TABLE
191
EGGS
I
Autopsy data on the occurrence of Heterakis gallinae and Histomonas meleagridis in chickens inoculated with: (I-l), Single Gravid Heterakis Containing Embryonated Eggs; (I-d), with Two Gravid Heterakis Embryonated Eggs; and (II), with Pooled Saline Suspension of Embryonated Eggs Obtained from about 100 Worms
-
Eggs fed
~-
Period after infection TOtal no.
8-15 days
18-56 days
Average Per bird -___
%
No.
740 76.0 529 47.2 914 47.6
2 I. 2
NO.
5 12
974 139 1120 224 1920 160
I-l I-2 II
39 8 48
6357 163 4025 63.3 1803 225 1485 82.3 7680 160 4665 60.7
-
-
I-
Eggs fed per case of histomoniasis
-
I-l I-2 II
7
Birds with Histomonas
WOl-lllS recovered
%
I-l
28.6 20.0 16.7
487
15 38.4 5 62.5 37 77.1
424
I-2
II
1120 960
361 208
received one worm each were positive for Histomonas. Among these birds, apparently one infection developed for each 424 embryonated eggs administered. Inasmuch as the whole worms that were fed and those opened to secure eggs for preparation of suspensions of eggs were all from the same source, the proportion of eggs that contained histomonads should have been similar. If that were true, the distribution of infected eggs in the various worms was not uniform, and the infected eggs were present in only half as many worms as random distribution would indicate. It follows then that the worms that did contain eggs infected with Histomonas must have averaged two “infective units” per worm. A minimum “infective unit” could presumably be one embryonated Heterakis egg containing one histomonad. Little or no relationship was observed between the number of embryonated eggs in a worm and its likelihood of having eggs containing Histomonas. Individual worms responsible for Histomonas infections recorded after the 18th day contained as few as 100 embryonated eggs and as many as 221, the average being 166. In the case of worms administered individually to birds that did not develop histomoniasis between the 18th and the 56th day, the average number of embryonated eggs per individual worm was 161; the number varied from 100 to 244. Worms with less than 100 embryonated eggs were not given individually.
192
EVERETT
E.
LUND
AND
ROY
H.
BURTNER,
JR.
The worms given in pairs contained an average of 112 embryonated eggs, and this was the same for birds that developed histomoniasis as for those that did not. If it is assumed, as the evidence indicates, that a worm containing 100 or 112 embryonated .eggs is as likely to carry Histomonas-infected eggs as one with 160 or 200 (one “infective unit” being all that is necessary to produce infection), further calculations are interesting. Administration of individual worms produced infections of Histomonas in 38.4 % of birds so inoculated (Table I). Theoretically, therefore, inoculation of birds with two worms containing embryonated eggs should result in 38.4 % + 38.4 % - (38.4%)2 = 76.8 % - 14.7 %, or 62.1% infection with Histomonas. This is in remarkably close agreement with the 62.5 % actually found (Table I). The one bird which received three worms containing a total of 179 embryonated eggs was positive for Histomonas, but the findings are considered to be of no value for statistical consideration and were therefore omitted from the calculations. Satisfactory numbers of cecal worms were recovered from the abovementioned histomonad-infected birds. In order to determine whether the eggs in the uteri of these worms contained viable histomonads, the following test was conducted. All the cecal worms recovered from a total of nine birds, autopsied 28, 33, and 35 days after experimental infection, were placed in saline in shallow culture dishes to induce development of the contained eggs. After development of the eggs was completed, the embryonated ones were collected and pooled, and 160 of them were administered to each of 60 susceptible turkey poults, 6 weeks of age. Forty of the birds so inoculated were killed for autopsy between the 18th and 57th day after inoculation. The birds in question are considered as being comparable to the chickens of Lot II (Table I). Twenty-eight (70 %) harbored infections of Histomonas at necropsy. One case of sub-clinical Histomonas infection occurred for each 228 embryonated eggs administered to the birds. This rate of infection with Histomonas was approximately the same as was obtained by the use of eggs from the parent stock of cecal worms. Tests carried out under different conditions have indicated that the number of viable Heterakis eggs required to produce a case of histomoniasis may be considerably less or vastly greater than was true for the material used for this study. In most cases, however, only a small
HISTOMONAD
INFECTIVITY
OF HETERAKIS
EGGS
193
proportion of the eggs contained viable histomonacls, as adjudged by the number of infections of this protozoan that were established in the susceptible test birds. SUMMARY
1. In tests herein reported, fewer than one of every 200 embryonatecl eggs of the cecal worm, Heterakis gallinae, were found to contain the blackhead parasite, Histomonas meleagriclis. This was determined by orally administering to chickens 6 weeks of age known numbers of cecal worm eggs which had been embryonated under laboratory conditions. 2. The observed rate of infectivity of the cecal worm eggs as regards Histomonas was approximately the same in eggs recovered from a succeeding generation of the worms, as determined by inoculation of susceptible birds with such eggs. 3. The number of Heterakis eggs containing Histomonas was not the same for all cecal worms. More than half of the worms contained no eggs with Histomonas, and worms which did contain such eggs averaged only two each. 4. These tests were conducted with a naturally-occuring, nonpathogenie strain of Histomonas. The situations herein described are not necessarily entirely comparable to those which exist with pathogenic strains.
EXPERIMENTAL
Infectivity with Everett Animal
Disease United
of Heterakis gallinue His tomonas meleagridis E. Lund
and Parasite
and
Roy
H. Burtner,
Research Branch, Agricultural of Agriculture, Beltsville,
States Department
(Submitted for publication,
Eggs Jr. Research Maryland
Service,
3 August 1956)
Histomonas meleagridis, the causative agent of blackhead in poultry, is easily maintained in eggs of the cecal worm, Heterakis gallinae, and blackhead disease is readily produced by the administration of the infected eggs of this nematode. Histomonads have not been discovered inside the cecal worm eggs, however, although a number of investigators have searched for them. Consequently, when an infection of blackhead is experimentally induced by feeding eggs of the cecal worm, the investigator knows only how many embryonated eggs he administers-not how many of them contain histomonads and therefore not how many Protozoa are introduced thereby. The investigation herein reported was conducted to determine what proportion of the eggs produced by cecal worms that develop in the alimentary tract of histomonad-infected birds may be expected to contain these protozoa. The method by which the determinations were made and the findings are summarized below. EXPERIMENTAL
PROCEDURE
Eggs of Heterakis gallinae which had been recovered from birds infected with a non-pathogenic strain of Histomonas were administered orally to three 5-week-old chickens. Histomonads were present in the cecal droppings of two of the inoculated birds from the 15th to the 39th day following the administration of eggs. Both birds were killed on the 41st day after infection. A total of 783 cecal worms, of which nearly 400 were gravid females, was recovered. The females were washed in several changes of 0.85 % salt solution and left intact in saline at room temperature for 16 days to permit the contained eggs to embryonate. At the end of that time about 50 % contained 189
190
EVERETT
E. LUND
AND
ROY H. BURTNER,
JR.
larvae, most of the remainder having failed to develop beyond the early stages of segmentation. New Hampshire Red chickens, 6-weeks-old, were divided into two lots of 60 each. The birds of one lot received whole worms. Those of the other received embryonated eggs taken from a large number of worms. All worms had been treated as described above. Forty-six birds of Lot I received one worm each. The smallest number of embryonated eggs in any worm administered to these birds was 100, the largest was 244, and the average was 160. Thirteen birds received two worms each. The worms selected for administration averaged 112 embryonated eggs each. Each bird received therefore approximately 224 such eggs. The remaining bird of Lot I received three worms, which contained a total of 179 embryonated eggs. In all cases the worms were introduced into the crop along with 1 ml of saline solution. Each of the 60 birds in Lot II received approximately 160 embryonated cecal worm eggs suspended in 1 ml of saline. These eggs were collected from about 100 worms selected from the same source as those given to the birds in Lot I. They were thoroughly mixed and suspended in saline, and the suspension was then calibrated by counting the number of embryonated eggs in 20 low-power fields of the microscope in each of eight 0.02-ml samples of the agitated suspension. Each sample filled the space under a cover glass 22-mm square. The average of the eight counts was then multiplied by a factor calculated to convert the figure to number of eggs per milliliter of suspension. The suspension was then diluted so that 1 ml contained approximately 160 embryonated eggs. Birds from each lot were killed at predesignated intervals from the 8th to the 56th day after infection and examined for both Histomcmas and Heterakis. OBSERVATIONS
AND
INTERPRETATIONS
The more important data are presented in the accompanying table. Because of the small number of birds that acquired infections of Histomonas, the examinations made during the first 2 weeks following experimental infection were not considered conclusive. For this reason, in the table the findings for this period are shown separately from the others. From the 18th day onward, 77 % of the chickens in Lot II were positive for Histomonas at autopsy. This is considered to represent one infection for each 208 embryonated eggs administered. For the corresponding period only 38.4 % of the chickens in Lot I that
HISTOMONAD
INFECTIVITY
OF HETERAKIS
TABLE
191
EGGS
I
Autopsy data on the occurrence of Heterakis gallinae and Histomonas meleagridis in chickens inoculated with: (I-l), Single Gravid Heterakis Containing Embryonated Eggs; (I-d), with Two Gravid Heterakis Embryonated Eggs; and (II), with Pooled Saline Suspension of Embryonated Eggs Obtained from about 100 Worms
-
Eggs fed
~-
Period after infection TOtal no.
8-15 days
18-56 days
Average Per bird -___
%
No.
740 76.0 529 47.2 914 47.6
2 I. 2
NO.
5 12
974 139 1120 224 1920 160
I-l I-2 II
39 8 48
6357 163 4025 63.3 1803 225 1485 82.3 7680 160 4665 60.7
-
-
I-
Eggs fed per case of histomoniasis
-
I-l I-2 II
7
Birds with Histomonas
WOl-lllS recovered
%
I-l
28.6 20.0 16.7
487
15 38.4 5 62.5 37 77.1
424
I-2
II
1120 960
361 208
received one worm each were positive for Histomonas. Among these birds, apparently one infection developed for each 424 embryonated eggs administered. Inasmuch as the whole worms that were fed and those opened to secure eggs for preparation of suspensions of eggs were all from the same source, the proportion of eggs that contained histomonads should have been similar. If that were true, the distribution of infected eggs in the various worms was not uniform, and the infected eggs were present in only half as many worms as random distribution would indicate. It follows then that the worms that did contain eggs infected with Histomonas must have averaged two “infective units” per worm. A minimum “infective unit” could presumably be one embryonated Heterakis egg containing one histomonad. Little or no relationship was observed between the number of embryonated eggs in a worm and its likelihood of having eggs containing Histomonas. Individual worms responsible for Histomonas infections recorded after the 18th day contained as few as 100 embryonated eggs and as many as 221, the average being 166. In the case of worms administered individually to birds that did not develop histomoniasis between the 18th and the 56th day, the average number of embryonated eggs per individual worm was 161; the number varied from 100 to 244. Worms with less than 100 embryonated eggs were not given individually.
192
EVERETT
E.
LUND
AND
ROY
H.
BURTNER,
JR.
The worms given in pairs contained an average of 112 embryonated eggs, and this was the same for birds that developed histomoniasis as for those that did not. If it is assumed, as the evidence indicates, that a worm containing 100 or 112 embryonated .eggs is as likely to carry Histomonas-infected eggs as one with 160 or 200 (one “infective unit” being all that is necessary to produce infection), further calculations are interesting. Administration of individual worms produced infections of Histomonas in 38.4 % of birds so inoculated (Table I). Theoretically, therefore, inoculation of birds with two worms containing embryonated eggs should result in 38.4 % + 38.4 % - (38.4%)2 = 76.8 % - 14.7 %, or 62.1% infection with Histomonas. This is in remarkably close agreement with the 62.5 % actually found (Table I). The one bird which received three worms containing a total of 179 embryonated eggs was positive for Histomonas, but the findings are considered to be of no value for statistical consideration and were therefore omitted from the calculations. Satisfactory numbers of cecal worms were recovered from the abovementioned histomonad-infected birds. In order to determine whether the eggs in the uteri of these worms contained viable histomonads, the following test was conducted. All the cecal worms recovered from a total of nine birds, autopsied 28, 33, and 35 days after experimental infection, were placed in saline in shallow culture dishes to induce development of the contained eggs. After development of the eggs was completed, the embryonated ones were collected and pooled, and 160 of them were administered to each of 60 susceptible turkey poults, 6 weeks of age. Forty of the birds so inoculated were killed for autopsy between the 18th and 57th day after inoculation. The birds in question are considered as being comparable to the chickens of Lot II (Table I). Twenty-eight (70 %) harbored infections of Histomonas at necropsy. One case of sub-clinical Histomonas infection occurred for each 228 embryonated eggs administered to the birds. This rate of infection with Histomonas was approximately the same as was obtained by the use of eggs from the parent stock of cecal worms. Tests carried out under different conditions have indicated that the number of viable Heterakis eggs required to produce a case of histomoniasis may be considerably less or vastly greater than was true for the material used for this study. In most cases, however, only a small
HISTOMONAD
INFECTIVITY
OF HETERAKIS
EGGS
193
proportion of the eggs contained viable histomonacls, as adjudged by the number of infections of this protozoan that were established in the susceptible test birds. SUMMARY
1. In tests herein reported, fewer than one of every 200 embryonatecl eggs of the cecal worm, Heterakis gallinae, were found to contain the blackhead parasite, Histomonas meleagriclis. This was determined by orally administering to chickens 6 weeks of age known numbers of cecal worm eggs which had been embryonated under laboratory conditions. 2. The observed rate of infectivity of the cecal worm eggs as regards Histomonas was approximately the same in eggs recovered from a succeeding generation of the worms, as determined by inoculation of susceptible birds with such eggs. 3. The number of Heterakis eggs containing Histomonas was not the same for all cecal worms. More than half of the worms contained no eggs with Histomonas, and worms which did contain such eggs averaged only two each. 4. These tests were conducted with a naturally-occuring, nonpathogenie strain of Histomonas. The situations herein described are not necessarily entirely comparable to those which exist with pathogenic strains.