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Larval Development of Cecal Worm (Heterakis gallinarum) in Chickens1
POLYUNSATURATES AND DYSTROPHY of nutritional dystrophy in the chick. Proc. Soc. Exp. Biol. Med. 109: 16-18. Leong, K. C , D. G. Snyder, G. M. Knobl and E. Gruger, 1962. Feeding of fish oil and ethyl ester fractions of fish oil to broilers. Poultry Sci. 4 1 : 1658. Leong, K. C , G. M. Knobl, Jr., D. G. Snyder and E. Gruger, 1964. Feeding of fish oil and ethyl ester fractions of fish oil to broilers. Poultry Sci. 43: 1235-1240. Machlin, L. J., and R. S. Gordon, 1960. Linoleic
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acid as causative agent of encephalomalacia in chickens fed oxidized fats. Proc. Soc. Exp. Biol. Med. 103: 659-663. Miller, D., K. C. Leong, G. M. Knobl, Jr. and E. H. Gruger, 1964. Exudative diathesis and muscular dystrophy in the chick induced by esters of polyunsaturated fatty acids. Proc. Soc. Exp. Biol. Med. 116: 1147-1151. Scott, M. L., 1957. The identification of unknown factor as selenium. Feedstuffs, 29: 20-21.
R. D. VATNE2 AND M. F. HANSEN Department of Zoology, Kansas State University, Manhattan (Received for publication January 6, 1965)
T
HE frequency of occurrence and significance of the temporary invasion of Heterakis gallinarum larvae into the cecal mucosa as part of the larvae's development is controversial. Riley and James (1921) reported marked inflammation of the cecal walls of infected chickens but did not describe a tissue phase. Dorman (1928) and Clapham (1933) indicated that all larvae developed in the lumen. Uribe (1922) designated the 2nd to the 5th day post-exposure as the period when all larvae were in the "bottom of the crypts of the cecal mucosa." According to Itagaki (1930) no larvae were found in the cecal wall but some were buried in 1
Contribution No. 341, Department of Zoology, Kansas State University, Manhattan, and Zoology Series No. 364. This study was supported in part by grant No. E-2124, National Institutes of Health and the Agricultural Experiment Station. 2 A portion of a dissertation presented by R. D. Vatne in partial fulfillment of the requirements for the degree Doctor of Philosophy in Parasitology at Kansas State University. Present address: Parasitology Section, Dr. Salsbury's Laboratories, Charles City, Iowa.
the cecal glands where they seemed to develop to maturity. Baker (1933) observed that "larvae showed a pronounced tendency to invade" but only the anterior one half of the larvae penetrated the glandular crypts of the mucosa. Tyzzer (1934) found the larvae penetrating the epithelia of cecal glands but none was found in underlying tissue. Although the 2nd to the Sth day was indicated as the usual period for tissue phase, some larvae returned to the lumen as early as the 2nd day. Roberts (1937) frequently observed larval penetration into the mucosa and occasionally into underlying reticular tissue. Hsu and Li (1940) occasionally saw larvae embedded in cecal mucosa 13 days post-exposure. Controversial tissue phase development of other species of nematode larvae was summarized by Madsen (1962) and Wehr and Hwang (1964). Terms used in the literature describing these larvae are tissue phase, histotropic phase, or dormant phase. The terms have been used to describe a variety of conditions which range from larvae lying in
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 24, 2015
Larval Development of Cecal Worm {Heterakis gallinarum) in Chickens1
1080
R. D. VATNE AND M. F. HANSEN
EXPERIMENTAL PROCEDURE
Because H. gallinarum is associated with the transmission of Histomonas meleagridis, a protozoan causing blackhead in fowl, some experimental chickens infected with H. gallinarum eggs developed cecal lesions of blackhead. Accordingly, results of experiments on the biology of that nematode had to be evaluated and designed to cope with the problem. Chickens showing cecal lesions of blackhead disease were not included. Lund (19S8) reported "Histomonas infections that do not provoke macroscopic cecal changes do not influence worm counts or worm lengths." Under the experimental conditions in our laboratory, macroscopic changes could usually be detected from the 9th to the 25th day post-exposure. All chickens used in the experiments were straight-run White Rocks received as day-old chicks from a commercial hatchery. They were intra-nasally vaccinated against Newcastle disease and infectious bronchitis, then maintained in heated
brooders for 2 weeks. One commercial ration was fed throughout the investigations. The preparation of standardized dosages of larvated ova for administration to the chickens was patterned after the sugar solution technique of Hansen et al. (195S). Adult heterakids were recovered by flushing cecal contents into quart jars. The jars were repeatedly filled with water and decanted until the worms were easily seen. Larvae were recovered from cecal tissues and lumina by using a modification of the Baermann technique. The ceca were slit longitudinally and the mucosal lining was gently but firmly scraped with a rubbertipped glass rod and then rinsed with water. Ceca were kept from curling by being placed between 2 circular screens of wire mesh, which were then stapled together. The screens were cut to fit about >4 inch from the top of a 3-inch funnel. The funnels were filled with tap water (41°C.) until the tissues (mucosal side down) were submerged. After incubation for 1 hour at 41 ° C , the larvae were drawn from the bottom of the funnel into Syracuse dishes and counted. Measurements were made of the first 10 lumen and tissue associated larvae in experiments 2 and 4. If fewer than 10 larvae were recovered from a bird, an average length of the worms from each bird in the group was determined and then a group average was calculated from the individual bird averages. The outlines of larvae as they appeared imposed on an ocular net micrometer were sketched on paper (8" by 8"), which was marked in a grid-like manner identical with the net micrometer. Worm outlines on the paper were traversed with a map measurer; the resulting distance was then reduced by the enlargement factor to obtain actual lengths. Representative sections of tissue from the small intestine and ceca were histologically examined to observe the relationship
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close proximity to the mucosa to the deep burrowing larvae in tissues. Frequently the terms are used without specific clarification, thus the association is obscure. This ambiguity may account for some of the discrepancies in the reported life history of H. gallinarum. Todd and Crowdus (19S2) described the larvae of Ascaridia galli lying in the mucus coating of the intestine as "intimately associated" with the tissue. From the results reported by the present authors, tissue association is appropos to most H. gallinarum larvae during the first few days of their life history. Because no quantitative studies have been reported on tissue association of H. gallinarum larvae, this study attempted to elucidate that point as well as the effects of numbers of larvae and age of the host on tissue association.
LARVAL DEVELOPMENT OF CECAL WORM
FIG. 1. Mean percentage of tissue associated larvae and days post-exposure
chickens 5, respectively. each group posure and exposure.
21, 49, and 77 days of age, Half of the chickens from were killed 4 days post-exthe other half, 7 days post-
RESULTS AND DISCUSSION
Experiment 1. The peak period of tissue association of the larvae with the ceca occurred the 3rd day following exposure (Fig. 1). A few larvae were found associated with the tissue for as long as 12 days post-exposure. The significance of tissue association is still a point of conjecture, however, it has frequently been described as a normal stage in development of the larvae. The temporary contact with tissue is regarded as a remnant of the behavioral pattern of certain related nematodes that migrate extensively through the body as an obligate part of their life history. Another view is that the tissue association is not a regular step in the normal development because not all larvae undergo tissue association, furthermore, they do not grow when embedded in tissue. Itagaki (1927) reported that the percentage of Ascaridia galli larvae closely associated with intestinal tissues varied according to the season of the year, suggesting that host resistance influenced their behavior. Madsen (1962) concluded that larval association with tissues "does not represent a phase in the
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of larvae to the mucosa. All tissues were fixed in Bouin's fluid, embedded in paraffin, sectioned at 12 microns, and stained with hematoxylin and eosin. Experiment 1. The percentage of larvae embedded in or in close association with the cecal lining was determined by examining the tissue and lumen contents of ceca from groups of chickens for 15 consecutive days post-exposure. Chickens, IS days of age, were infected with 200 ( ± 20) larvated Heterakis eggs. Six chickens were necropsied on consecutive days post-exposure. Beginning on the 10th day, it was necessary to necropsy 7 to 9 chickens daily to obtain 6 without blackhead lesions. On the 15th day only 4 chickens were necropsied. Histological sections of the ceca and the small intestine were made and examined from chickens infected for 1, 3, and 5 days, respectively. Experiment 2. A comparative study of tissue associated larvae was made between larvae that developed in chickens infected at 10 days of age and those infected at 35 days of age. Five chickens from each age group were examined daily for the first 7 days post-exposure. Starting with the 8th day post-exposure some ceca showed evidence of blackhead disease, thus a larger number of chickens were examined each day to find 5 or fewer not affected by the disease. Experiment 3. Forty-three chickens were examined to determine the cecal habitat (distal Yz, medial }i, proximal Y lumen or tissue associated, respectively) of Heterakis adults or larvae at periods ranging from 3 to 85 days post-exposure. Experiment 4. Because 10-day-old chickens were more resistant than 35-day-old birds to infection with H. galUnarum, an experiment was designed to investigate the influence of host's age on the development of the nematode. Larvated eggs (200 ± 20) were administered to 4 groups of
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R. D. VATNE AND M. F. HANSEN
IO-DAY-OLD CHICKENS 35-DAY-OLD CHICKENS
NUMBER
OF
DAYS
sense of a behavioral pattern innate to the parasite, but rather a phenomenon on the degree of resistance in the host." Histological sections of ceca from chickens 1, 3, and S days post-exposure showed that most larvae were located in the crypts of the mucosa, although a few were observed in interstitial or muscular tissue. Penetration into the mucosa of only the
I IO-DAY-OLD
CHICKENS
• 35-DAY-OLD
CHICKENS
IO-DAY-OLD 35-0AY-OL0
\ \ \
J
3
\ >
L
i_l
l L 2
4
5 NUMBER
CHICKENS CHICKENS
6
7 OF
8
J
DAYS
FIG. 3. Length of larvae recovered from the cecal lumen and days post-exposure.
' 2
3
I 4
1' I 5
NUMBER
6 OF
1 7
8
9
(
0
)
1
DAYS
FIG. 4. Length of tissue associated larvae and days post-exposure.
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FIG. 2. Percentage of tissue associated larvae with respect to the age of chickens at exposure and days post-exposure.
anterior portion of the larvae was rarely observed. Apparently the only damage to the cecal tissue was a mechanical displacement of cells. Histological sections of the small intestinal wall were negative for larvae. Experiment 2. Larvae developing in the group of chickens infected at 35 days of age demonstrated greater affinity for cecal tissues up to the Sth day post-exposure than larvae recovered from chickens infected at 10 days of age (Fig. 2). No statistically significant differences were noted in the rate of growth of lumen and tissue associated larvae, respectively, betweeen age groups of chickens (Figs. 3, 4). A few diminutive larvae were recovered from the cecal tissue and the lumen (Fig. 4, 10 days). Presumably, the larvae recovered from the lumen had penetrated into cecal tissues (true tissue phase) and had recently returned to the lumen when recovered at necropsy. Also, there was no difference in the growth rate between tissue and lumen
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LARVAL DEVELOPMENT OF CECAL WORM
TABLE 1.—Distribution of lumen larvae in the ceca Percentage of larvae ChickEggs Postens administered exposure Proximal Medial i Distal J (no.) (no.) (hours)
i
5 5 5 5 5 5 5 5
100 1,000 100 500 100 100 500 1,000
72 75 90 94 96 96 98 99
7.5 0.9 1.1 4.6 0.8 5.6 1.9 2.1
75.0 77.5 72.9 59.4 69.4 62.0 63.5 67.1
17.5 21.6 26.0 36.0 29.8 32.4 34.6 30.8
TABLE
2.-
-Distribulion of tissue associated larvae in the ceca
Eggs Percentage of larvae PostChickens Adminis- exposure (no.) tered Proximal (hours) Medial J Distal i (no.)
i
5 5 5 5 6 3 2 2
100 500 100 500 100 100 500 1,000
90 94 96 98 240 408 576 2,040
3.4 0 0 1.2 0.6 5.9 0 0
53.3 37.8 48.0 61.3 23.7 22.4 14.0 23.0
43.3 62.2 52.0 37.5 75.7 71.7 86.0 77.0
that the majority of heterakids had moved to the apices of the ceca by the 17th day post-exposure. Larvae of Heterakis bonosae from bobwhite quail were reported to burrow temporarily into the mucosa at the ileocecal junctions (Ward, 1947). The number of larvated eggs administered (100-1000) did not influence the distribution of lumen or tissue associated larvae (Tables 1, 2). Because of the relationship between Heterakis and Histomonas meleagridis", knowledge of the cecal area where larvae are localized during tissue association is important. For example, studies on H. meleagridis subsequent to administration of cecal worm eggs, may now be expedited by searching for the histomonads in the medial portion of the ceca. Lund (1959) theorized that the penetration by heterakid larvae into the cecal linings of chickens previously immunized with a nonpathogenic strain of histomonads offered them a means of passing beyond the immune barrier of the mucosa. Further studies on this theory, as well as studies on the effects of anthelmintics on heterakids during their tissue association, may be enhanced by knowledge of infection sites demonstrated in this study. Experiment 4. The percentage of tissue associated larvae was larger in birds 21 days or older than in 5-day-old birds (Tables 3, 4). The data complement those reported in experiment 2 that larvae in older chickens were more closely associated with
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larvae and within a 3-day growth period both types of larvae had doubled in length (Tables 3, 4). Those data support the contention that H. gallinarum does not have, or rarely has, a true tissue phase in its life cycle. Fewer larvae were recovered from the chickens infected at 10 days of age (1133) than from those infected at 35 days of age (1700). The larger number of larvae and greater tissue association among 35day old birds (Fig. 2) parallels susceptibility of fowl to histomoniasis. Ohara and Reid (1961) reported that chickens 21 to 56 days of age were more susceptible to Histomonas meleagridis infections than younger or older birds. Desowitz (1951) reported that chickens were less susceptible at 6 days of age than at 21 days. The difference in age susceptibility of chickens to heterakids is not readily explainable. Possibly the cecal flora in chicks 10 days of age provide a less suitable habitat for heterakids than in older birds. Suomalainen and Arhimo (1945) reported that cellulose decomposing activity was less in cultures of cecal bacteria from young wild gallinaceous birds than from adults. Experiment 3. During the period, to 98 hours post-exposure, of close association with the tissues, the majority of heterakids inhabited the medial J/z of the ceca, whereas the majority inhabited the distal }i at 240 hours post-exposure (Table 2). The results are in accord with those reported by Grigorev (1959) even though he stated
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R. D. VATNE AND M. F. HANSEN
TABLE 3.—Comparison of larvae recovered from groups (10 chickens each) infected at different ages. Necropsied 4 days post-exposure Age of groups at exposure (days) 5 21 49 77
Tissue associated larve
Lumen larvae
Number (avg.)
Avg. length (mm.)
% of total larvae recovered
Number (avg.)
17.4 23.0 21.8 15.0
.69 .70 .72 .72
57.7 76.2 85.2 90.1
16.0 7.2 3.8 1.5
TABLE 4.-
.70 .76 .70 .72
,,, , , total avg. - > - - / 33.4 30.2 25.6 16.5
that larvae recovered from birds exposed at 21 days of age grew more rapidly than those recovered from 5-day-old birds. Comparison of those results with results reported in experiment 2 shows that repression of growth of lumen larvae occurs in birds at some age less than 10 days old. With numbers and growth rate of lumen larvae as criteria of host susceptibility, the 21-day-old birds were the most susceptible to H. gallinarum. SUMMARY
Larval development of Heterakis gallinarum in chickens was quantitatively studied. Larvae were found in close association or occasionally embedded in cecal tissues (medial Yi of cecum) until 12 days post-exposure. The peak association was 3 days post-exposure. By the 10th day postexposure most of the larvae had returned to the lumen and were in the distal Yi of the ceca. More larvae were associated with cecal tissues in birds 21 days or older than in 5-day-old birds. Tissue association tended
-Comparison of larvae recovered from groups (10 chickens each) infected at d\ Necropsied 7 days post-exposure
ages.
Lumen larvae
Tissue associated larvae
Age of groups at exposure (days)
Number (avg.)
Avg. length (mm.)
% of total larvae recovered
Number (avg.)
5 21 49 77
0.5 2.2 1.6 0.8
1.45 1.59 1.57 1.81
1.9 6.4 6.2 4.8
25.6 32.0 24.2 17.4
Total avg. no. Avg. length larvae/chicken (mm.) 1.45 1.54 1.48 1.63
26.1 34.2 25.8 18.3
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cecal tissues up to 4 days post-exposure. Age of birds at exposure did not influence the growth rate, as reflected in linear length, of tissue associated larvae among chickens necropsied 4 days post-exposure (Table 3). Only small numbers of tissue associated larvae were recovered from chickens necropsied 7 days post-exposure, as by then many of them had returned to the lumen (Table 4). However, those found associated with tissue were about twice as large as those recovered from chickens necropsied 3 days earlier (Table 3). The total average number of larvae per chicken (tissue and lumen) recovered from groups examined 4 days post-exposure decreased with increased age of chickens at exposure (P < 0.05) (Table 3). Among chickens necropsied 7 days post-exposure (Table 4), the 77-day-old group harbored fewer (P < 0.05) larvae than the group exposed at 21 days of age (L.S.D. = 11.75) (Table 4). Statistical analyses of growth rates of lumen larvae given in Tables 3 and 4 show
Avg. length (mm.)
LARVAL DEVELOPMENT OF CECAL WORM
REFERENCES Baker, A. D., 1933. Some observations on ths development of the cecal worm. Heterakis gallinae (Gmelin, 1790) Freeborn, 1923, in the domestic fowl. Sci. Agr. 13 : 356-363. Clapham, P. A., 1933. On the life history of Heterakis gallinae. J. Helminthol. 11: 67-86. Desowitz, R. S., 1951. Age as a factor influencing fatal injections of histomoniasis in chickens. J. Comp. Path. Therap. 6 1 : 231-236. Dorman, H. P., 1928. Studies on the life cycle of Heterakis papulosa Bloch. Trans. Am. Microscop. Soc. 47: 379-413. Grigorev, N. K., 1959. Pathological changes in the caecum and liver of domestic fowls infected with Heterakis. Trudy Moskovskoe Vet. Akad. 25: 245-254. Hansen, M. F., C. J. Terhaar and D. S. Turner, 1955. Importance of the egg shell of Ascaridia galli to the infectivity of its larva. J. Parasitol. 42: 122-125. Hsu, H. F., and S. Y. Li, 1940. Studies on the food and the digestive system of certain parasites. VI. On the food of certain helminths living in the digestive tract of vertebrates. Chin. Med. J. 57: 559-569. Itagaki, S., 1930. The nature of the parasitic nodules in the caecal wall of fowls and the
development of Heterakis vesicularis. Rep. Proc. World's Poul. Cong. (London, England) pp. 517-520. Lund, E. E., 1958. Growth and development of Heterakis gallinae in turkeys and chickens infected with Histomonas meleagridis. J. Parasitol. 44: 297-301. Lund, E. E., 1959. Factors influencing the distribution of Heterakis in the ceca of chickens and turkeys. J. Parasitol. 45 (Sec. 2 ) : 43. Madsen, H., 1962. On the interaction between Heterakis gallinarum, Ascardia galli, "blackhead" and the chicken. J. Helminthol. 36: 107-142. Ohara, T., and W. M. Reid, 1961. Histomoniasis in chickens: age of greatest susceptibility and pathogenicity studies. Avian Dis. 5: 355-361. Riley, W. A., and L. G. James, 1921. Studies on the chicken nematode, Heterakis papulosa (Bloch). J. Am. Vet. Med. Assoc. 22: 208-217. 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. Austral. J. Exp. Biol. Med. Sci. 15:429-439. Soumalainen, H., and E. Arhimo, 1945. On the microbial decomposition of cellulose by wild gallinaceous birds (family: Tetraonidae). Ornis Fennica, 22: 21-23. Todd, A. C , and D. H. Crowdus, 1952. On the life history of Ascaridia galli. Trans. Am. Microscop. Soc. 71: 282-287. Tyzzer, E. E., 1934. Studies on histomoniasis, or "blackhead" infection, in the chicken and the turkey. Proc. Am. Acad. Arts Sci. 69: 189-264. Uribe, C , 1922. Observations on the development of Heterakis papulosa Bloch in the chicken. J. Parasitol. 8: 167-176. Ward, J. W., 1947. The life history and bionomics of Heterakis bonasae a cecal nematode of bobwhite quail, Colinus virginianus and C. virginianus texanus. J. Parasitol. 33(Sec. 2 ) : 23. Wehr, E. E., and J. C. Hwang, 1964. The life cycle and morphology of Ascaridia columbae. (Gmelin, 1790) Travassos, 1913 (Nematoda: Ascarididae) in the domestic pigeon {Columba livia domestica). J. Parasitol. 50: 131-137.
FEBRUARY 11-13. FACT FINDING CONFERENCE, INSTITUTE OF AMERICAN POULTRY INDUSTRIES, MUNICIPAL AUDITORIUM, KANSAS CITY. ' AUGUST 3-10, 1966. SEVENTH INTERNATIONAL CONGRESS OF NUTRITION, HAMBURG, GERMANY
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to increase with age of birds. Age of birds did not influence growth rate of tissue associated larvae, whereas, growth rate of lumen larvae was repressed in birds at some age less than 10 days old. Because tissue associated larvae and lumen larvae grew at comparable rates and only occasionally were larvae observed to penetrate the cecal mucosa, a true tissue phase rarely occurs in the life cycle of H. gallinarum. With numbers and growth rate of lumen larvae as criteria of host susceptibility, 21-day-old chickens were the most susceptible to H. gallinarum.
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1079
acid as causative agent of encephalomalacia in chickens fed oxidized fats. Proc. Soc. Exp. Biol. Med. 103: 659-663. Miller, D., K. C. Leong, G. M. Knobl, Jr. and E. H. Gruger, 1964. Exudative diathesis and muscular dystrophy in the chick induced by esters of polyunsaturated fatty acids. Proc. Soc. Exp. Biol. Med. 116: 1147-1151. Scott, M. L., 1957. The identification of unknown factor as selenium. Feedstuffs, 29: 20-21.
R. D. VATNE2 AND M. F. HANSEN Department of Zoology, Kansas State University, Manhattan (Received for publication January 6, 1965)
T
HE frequency of occurrence and significance of the temporary invasion of Heterakis gallinarum larvae into the cecal mucosa as part of the larvae's development is controversial. Riley and James (1921) reported marked inflammation of the cecal walls of infected chickens but did not describe a tissue phase. Dorman (1928) and Clapham (1933) indicated that all larvae developed in the lumen. Uribe (1922) designated the 2nd to the 5th day post-exposure as the period when all larvae were in the "bottom of the crypts of the cecal mucosa." According to Itagaki (1930) no larvae were found in the cecal wall but some were buried in 1
Contribution No. 341, Department of Zoology, Kansas State University, Manhattan, and Zoology Series No. 364. This study was supported in part by grant No. E-2124, National Institutes of Health and the Agricultural Experiment Station. 2 A portion of a dissertation presented by R. D. Vatne in partial fulfillment of the requirements for the degree Doctor of Philosophy in Parasitology at Kansas State University. Present address: Parasitology Section, Dr. Salsbury's Laboratories, Charles City, Iowa.
the cecal glands where they seemed to develop to maturity. Baker (1933) observed that "larvae showed a pronounced tendency to invade" but only the anterior one half of the larvae penetrated the glandular crypts of the mucosa. Tyzzer (1934) found the larvae penetrating the epithelia of cecal glands but none was found in underlying tissue. Although the 2nd to the Sth day was indicated as the usual period for tissue phase, some larvae returned to the lumen as early as the 2nd day. Roberts (1937) frequently observed larval penetration into the mucosa and occasionally into underlying reticular tissue. Hsu and Li (1940) occasionally saw larvae embedded in cecal mucosa 13 days post-exposure. Controversial tissue phase development of other species of nematode larvae was summarized by Madsen (1962) and Wehr and Hwang (1964). Terms used in the literature describing these larvae are tissue phase, histotropic phase, or dormant phase. The terms have been used to describe a variety of conditions which range from larvae lying in
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 24, 2015
Larval Development of Cecal Worm {Heterakis gallinarum) in Chickens1
1080
R. D. VATNE AND M. F. HANSEN
EXPERIMENTAL PROCEDURE
Because H. gallinarum is associated with the transmission of Histomonas meleagridis, a protozoan causing blackhead in fowl, some experimental chickens infected with H. gallinarum eggs developed cecal lesions of blackhead. Accordingly, results of experiments on the biology of that nematode had to be evaluated and designed to cope with the problem. Chickens showing cecal lesions of blackhead disease were not included. Lund (19S8) reported "Histomonas infections that do not provoke macroscopic cecal changes do not influence worm counts or worm lengths." Under the experimental conditions in our laboratory, macroscopic changes could usually be detected from the 9th to the 25th day post-exposure. All chickens used in the experiments were straight-run White Rocks received as day-old chicks from a commercial hatchery. They were intra-nasally vaccinated against Newcastle disease and infectious bronchitis, then maintained in heated
brooders for 2 weeks. One commercial ration was fed throughout the investigations. The preparation of standardized dosages of larvated ova for administration to the chickens was patterned after the sugar solution technique of Hansen et al. (195S). Adult heterakids were recovered by flushing cecal contents into quart jars. The jars were repeatedly filled with water and decanted until the worms were easily seen. Larvae were recovered from cecal tissues and lumina by using a modification of the Baermann technique. The ceca were slit longitudinally and the mucosal lining was gently but firmly scraped with a rubbertipped glass rod and then rinsed with water. Ceca were kept from curling by being placed between 2 circular screens of wire mesh, which were then stapled together. The screens were cut to fit about >4 inch from the top of a 3-inch funnel. The funnels were filled with tap water (41°C.) until the tissues (mucosal side down) were submerged. After incubation for 1 hour at 41 ° C , the larvae were drawn from the bottom of the funnel into Syracuse dishes and counted. Measurements were made of the first 10 lumen and tissue associated larvae in experiments 2 and 4. If fewer than 10 larvae were recovered from a bird, an average length of the worms from each bird in the group was determined and then a group average was calculated from the individual bird averages. The outlines of larvae as they appeared imposed on an ocular net micrometer were sketched on paper (8" by 8"), which was marked in a grid-like manner identical with the net micrometer. Worm outlines on the paper were traversed with a map measurer; the resulting distance was then reduced by the enlargement factor to obtain actual lengths. Representative sections of tissue from the small intestine and ceca were histologically examined to observe the relationship
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 24, 2015
close proximity to the mucosa to the deep burrowing larvae in tissues. Frequently the terms are used without specific clarification, thus the association is obscure. This ambiguity may account for some of the discrepancies in the reported life history of H. gallinarum. Todd and Crowdus (19S2) described the larvae of Ascaridia galli lying in the mucus coating of the intestine as "intimately associated" with the tissue. From the results reported by the present authors, tissue association is appropos to most H. gallinarum larvae during the first few days of their life history. Because no quantitative studies have been reported on tissue association of H. gallinarum larvae, this study attempted to elucidate that point as well as the effects of numbers of larvae and age of the host on tissue association.
LARVAL DEVELOPMENT OF CECAL WORM
FIG. 1. Mean percentage of tissue associated larvae and days post-exposure
chickens 5, respectively. each group posure and exposure.
21, 49, and 77 days of age, Half of the chickens from were killed 4 days post-exthe other half, 7 days post-
RESULTS AND DISCUSSION
Experiment 1. The peak period of tissue association of the larvae with the ceca occurred the 3rd day following exposure (Fig. 1). A few larvae were found associated with the tissue for as long as 12 days post-exposure. The significance of tissue association is still a point of conjecture, however, it has frequently been described as a normal stage in development of the larvae. The temporary contact with tissue is regarded as a remnant of the behavioral pattern of certain related nematodes that migrate extensively through the body as an obligate part of their life history. Another view is that the tissue association is not a regular step in the normal development because not all larvae undergo tissue association, furthermore, they do not grow when embedded in tissue. Itagaki (1927) reported that the percentage of Ascaridia galli larvae closely associated with intestinal tissues varied according to the season of the year, suggesting that host resistance influenced their behavior. Madsen (1962) concluded that larval association with tissues "does not represent a phase in the
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 24, 2015
of larvae to the mucosa. All tissues were fixed in Bouin's fluid, embedded in paraffin, sectioned at 12 microns, and stained with hematoxylin and eosin. Experiment 1. The percentage of larvae embedded in or in close association with the cecal lining was determined by examining the tissue and lumen contents of ceca from groups of chickens for 15 consecutive days post-exposure. Chickens, IS days of age, were infected with 200 ( ± 20) larvated Heterakis eggs. Six chickens were necropsied on consecutive days post-exposure. Beginning on the 10th day, it was necessary to necropsy 7 to 9 chickens daily to obtain 6 without blackhead lesions. On the 15th day only 4 chickens were necropsied. Histological sections of the ceca and the small intestine were made and examined from chickens infected for 1, 3, and 5 days, respectively. Experiment 2. A comparative study of tissue associated larvae was made between larvae that developed in chickens infected at 10 days of age and those infected at 35 days of age. Five chickens from each age group were examined daily for the first 7 days post-exposure. Starting with the 8th day post-exposure some ceca showed evidence of blackhead disease, thus a larger number of chickens were examined each day to find 5 or fewer not affected by the disease. Experiment 3. Forty-three chickens were examined to determine the cecal habitat (distal Yz, medial }i, proximal Y lumen or tissue associated, respectively) of Heterakis adults or larvae at periods ranging from 3 to 85 days post-exposure. Experiment 4. Because 10-day-old chickens were more resistant than 35-day-old birds to infection with H. galUnarum, an experiment was designed to investigate the influence of host's age on the development of the nematode. Larvated eggs (200 ± 20) were administered to 4 groups of
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IO-DAY-OLD CHICKENS 35-DAY-OLD CHICKENS
NUMBER
OF
DAYS
sense of a behavioral pattern innate to the parasite, but rather a phenomenon on the degree of resistance in the host." Histological sections of ceca from chickens 1, 3, and S days post-exposure showed that most larvae were located in the crypts of the mucosa, although a few were observed in interstitial or muscular tissue. Penetration into the mucosa of only the
I IO-DAY-OLD
CHICKENS
• 35-DAY-OLD
CHICKENS
IO-DAY-OLD 35-0AY-OL0
\ \ \
J
3
\ >
L
i_l
l L 2
4
5 NUMBER
CHICKENS CHICKENS
6
7 OF
8
J
DAYS
FIG. 3. Length of larvae recovered from the cecal lumen and days post-exposure.
' 2
3
I 4
1' I 5
NUMBER
6 OF
1 7
8
9
(
0
)
1
DAYS
FIG. 4. Length of tissue associated larvae and days post-exposure.
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FIG. 2. Percentage of tissue associated larvae with respect to the age of chickens at exposure and days post-exposure.
anterior portion of the larvae was rarely observed. Apparently the only damage to the cecal tissue was a mechanical displacement of cells. Histological sections of the small intestinal wall were negative for larvae. Experiment 2. Larvae developing in the group of chickens infected at 35 days of age demonstrated greater affinity for cecal tissues up to the Sth day post-exposure than larvae recovered from chickens infected at 10 days of age (Fig. 2). No statistically significant differences were noted in the rate of growth of lumen and tissue associated larvae, respectively, betweeen age groups of chickens (Figs. 3, 4). A few diminutive larvae were recovered from the cecal tissue and the lumen (Fig. 4, 10 days). Presumably, the larvae recovered from the lumen had penetrated into cecal tissues (true tissue phase) and had recently returned to the lumen when recovered at necropsy. Also, there was no difference in the growth rate between tissue and lumen
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LARVAL DEVELOPMENT OF CECAL WORM
TABLE 1.—Distribution of lumen larvae in the ceca Percentage of larvae ChickEggs Postens administered exposure Proximal Medial i Distal J (no.) (no.) (hours)
i
5 5 5 5 5 5 5 5
100 1,000 100 500 100 100 500 1,000
72 75 90 94 96 96 98 99
7.5 0.9 1.1 4.6 0.8 5.6 1.9 2.1
75.0 77.5 72.9 59.4 69.4 62.0 63.5 67.1
17.5 21.6 26.0 36.0 29.8 32.4 34.6 30.8
TABLE
2.-
-Distribulion of tissue associated larvae in the ceca
Eggs Percentage of larvae PostChickens Adminis- exposure (no.) tered Proximal (hours) Medial J Distal i (no.)
i
5 5 5 5 6 3 2 2
100 500 100 500 100 100 500 1,000
90 94 96 98 240 408 576 2,040
3.4 0 0 1.2 0.6 5.9 0 0
53.3 37.8 48.0 61.3 23.7 22.4 14.0 23.0
43.3 62.2 52.0 37.5 75.7 71.7 86.0 77.0
that the majority of heterakids had moved to the apices of the ceca by the 17th day post-exposure. Larvae of Heterakis bonosae from bobwhite quail were reported to burrow temporarily into the mucosa at the ileocecal junctions (Ward, 1947). The number of larvated eggs administered (100-1000) did not influence the distribution of lumen or tissue associated larvae (Tables 1, 2). Because of the relationship between Heterakis and Histomonas meleagridis", knowledge of the cecal area where larvae are localized during tissue association is important. For example, studies on H. meleagridis subsequent to administration of cecal worm eggs, may now be expedited by searching for the histomonads in the medial portion of the ceca. Lund (1959) theorized that the penetration by heterakid larvae into the cecal linings of chickens previously immunized with a nonpathogenic strain of histomonads offered them a means of passing beyond the immune barrier of the mucosa. Further studies on this theory, as well as studies on the effects of anthelmintics on heterakids during their tissue association, may be enhanced by knowledge of infection sites demonstrated in this study. Experiment 4. The percentage of tissue associated larvae was larger in birds 21 days or older than in 5-day-old birds (Tables 3, 4). The data complement those reported in experiment 2 that larvae in older chickens were more closely associated with
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larvae and within a 3-day growth period both types of larvae had doubled in length (Tables 3, 4). Those data support the contention that H. gallinarum does not have, or rarely has, a true tissue phase in its life cycle. Fewer larvae were recovered from the chickens infected at 10 days of age (1133) than from those infected at 35 days of age (1700). The larger number of larvae and greater tissue association among 35day old birds (Fig. 2) parallels susceptibility of fowl to histomoniasis. Ohara and Reid (1961) reported that chickens 21 to 56 days of age were more susceptible to Histomonas meleagridis infections than younger or older birds. Desowitz (1951) reported that chickens were less susceptible at 6 days of age than at 21 days. The difference in age susceptibility of chickens to heterakids is not readily explainable. Possibly the cecal flora in chicks 10 days of age provide a less suitable habitat for heterakids than in older birds. Suomalainen and Arhimo (1945) reported that cellulose decomposing activity was less in cultures of cecal bacteria from young wild gallinaceous birds than from adults. Experiment 3. During the period, to 98 hours post-exposure, of close association with the tissues, the majority of heterakids inhabited the medial J/z of the ceca, whereas the majority inhabited the distal }i at 240 hours post-exposure (Table 2). The results are in accord with those reported by Grigorev (1959) even though he stated
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R. D. VATNE AND M. F. HANSEN
TABLE 3.—Comparison of larvae recovered from groups (10 chickens each) infected at different ages. Necropsied 4 days post-exposure Age of groups at exposure (days) 5 21 49 77
Tissue associated larve
Lumen larvae
Number (avg.)
Avg. length (mm.)
% of total larvae recovered
Number (avg.)
17.4 23.0 21.8 15.0
.69 .70 .72 .72
57.7 76.2 85.2 90.1
16.0 7.2 3.8 1.5
TABLE 4.-
.70 .76 .70 .72
,,, , , total avg. - > - - / 33.4 30.2 25.6 16.5
that larvae recovered from birds exposed at 21 days of age grew more rapidly than those recovered from 5-day-old birds. Comparison of those results with results reported in experiment 2 shows that repression of growth of lumen larvae occurs in birds at some age less than 10 days old. With numbers and growth rate of lumen larvae as criteria of host susceptibility, the 21-day-old birds were the most susceptible to H. gallinarum. SUMMARY
Larval development of Heterakis gallinarum in chickens was quantitatively studied. Larvae were found in close association or occasionally embedded in cecal tissues (medial Yi of cecum) until 12 days post-exposure. The peak association was 3 days post-exposure. By the 10th day postexposure most of the larvae had returned to the lumen and were in the distal Yi of the ceca. More larvae were associated with cecal tissues in birds 21 days or older than in 5-day-old birds. Tissue association tended
-Comparison of larvae recovered from groups (10 chickens each) infected at d\ Necropsied 7 days post-exposure
ages.
Lumen larvae
Tissue associated larvae
Age of groups at exposure (days)
Number (avg.)
Avg. length (mm.)
% of total larvae recovered
Number (avg.)
5 21 49 77
0.5 2.2 1.6 0.8
1.45 1.59 1.57 1.81
1.9 6.4 6.2 4.8
25.6 32.0 24.2 17.4
Total avg. no. Avg. length larvae/chicken (mm.) 1.45 1.54 1.48 1.63
26.1 34.2 25.8 18.3
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cecal tissues up to 4 days post-exposure. Age of birds at exposure did not influence the growth rate, as reflected in linear length, of tissue associated larvae among chickens necropsied 4 days post-exposure (Table 3). Only small numbers of tissue associated larvae were recovered from chickens necropsied 7 days post-exposure, as by then many of them had returned to the lumen (Table 4). However, those found associated with tissue were about twice as large as those recovered from chickens necropsied 3 days earlier (Table 3). The total average number of larvae per chicken (tissue and lumen) recovered from groups examined 4 days post-exposure decreased with increased age of chickens at exposure (P < 0.05) (Table 3). Among chickens necropsied 7 days post-exposure (Table 4), the 77-day-old group harbored fewer (P < 0.05) larvae than the group exposed at 21 days of age (L.S.D. = 11.75) (Table 4). Statistical analyses of growth rates of lumen larvae given in Tables 3 and 4 show
Avg. length (mm.)
LARVAL DEVELOPMENT OF CECAL WORM
REFERENCES Baker, A. D., 1933. Some observations on ths development of the cecal worm. Heterakis gallinae (Gmelin, 1790) Freeborn, 1923, in the domestic fowl. Sci. Agr. 13 : 356-363. Clapham, P. A., 1933. On the life history of Heterakis gallinae. J. Helminthol. 11: 67-86. Desowitz, R. S., 1951. Age as a factor influencing fatal injections of histomoniasis in chickens. J. Comp. Path. Therap. 6 1 : 231-236. Dorman, H. P., 1928. Studies on the life cycle of Heterakis papulosa Bloch. Trans. Am. Microscop. Soc. 47: 379-413. Grigorev, N. K., 1959. Pathological changes in the caecum and liver of domestic fowls infected with Heterakis. Trudy Moskovskoe Vet. Akad. 25: 245-254. Hansen, M. F., C. J. Terhaar and D. S. Turner, 1955. Importance of the egg shell of Ascaridia galli to the infectivity of its larva. J. Parasitol. 42: 122-125. Hsu, H. F., and S. Y. Li, 1940. Studies on the food and the digestive system of certain parasites. VI. On the food of certain helminths living in the digestive tract of vertebrates. Chin. Med. J. 57: 559-569. Itagaki, S., 1930. The nature of the parasitic nodules in the caecal wall of fowls and the
development of Heterakis vesicularis. Rep. Proc. World's Poul. Cong. (London, England) pp. 517-520. Lund, E. E., 1958. Growth and development of Heterakis gallinae in turkeys and chickens infected with Histomonas meleagridis. J. Parasitol. 44: 297-301. Lund, E. E., 1959. Factors influencing the distribution of Heterakis in the ceca of chickens and turkeys. J. Parasitol. 45 (Sec. 2 ) : 43. Madsen, H., 1962. On the interaction between Heterakis gallinarum, Ascardia galli, "blackhead" and the chicken. J. Helminthol. 36: 107-142. Ohara, T., and W. M. Reid, 1961. Histomoniasis in chickens: age of greatest susceptibility and pathogenicity studies. Avian Dis. 5: 355-361. Riley, W. A., and L. G. James, 1921. Studies on the chicken nematode, Heterakis papulosa (Bloch). J. Am. Vet. Med. Assoc. 22: 208-217. 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. Austral. J. Exp. Biol. Med. Sci. 15:429-439. Soumalainen, H., and E. Arhimo, 1945. On the microbial decomposition of cellulose by wild gallinaceous birds (family: Tetraonidae). Ornis Fennica, 22: 21-23. Todd, A. C , and D. H. Crowdus, 1952. On the life history of Ascaridia galli. Trans. Am. Microscop. Soc. 71: 282-287. Tyzzer, E. E., 1934. Studies on histomoniasis, or "blackhead" infection, in the chicken and the turkey. Proc. Am. Acad. Arts Sci. 69: 189-264. Uribe, C , 1922. Observations on the development of Heterakis papulosa Bloch in the chicken. J. Parasitol. 8: 167-176. Ward, J. W., 1947. The life history and bionomics of Heterakis bonasae a cecal nematode of bobwhite quail, Colinus virginianus and C. virginianus texanus. J. Parasitol. 33(Sec. 2 ) : 23. Wehr, E. E., and J. C. Hwang, 1964. The life cycle and morphology of Ascaridia columbae. (Gmelin, 1790) Travassos, 1913 (Nematoda: Ascarididae) in the domestic pigeon {Columba livia domestica). J. Parasitol. 50: 131-137.
FEBRUARY 11-13. FACT FINDING CONFERENCE, INSTITUTE OF AMERICAN POULTRY INDUSTRIES, MUNICIPAL AUDITORIUM, KANSAS CITY. ' AUGUST 3-10, 1966. SEVENTH INTERNATIONAL CONGRESS OF NUTRITION, HAMBURG, GERMANY
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to increase with age of birds. Age of birds did not influence growth rate of tissue associated larvae, whereas, growth rate of lumen larvae was repressed in birds at some age less than 10 days old. Because tissue associated larvae and lumen larvae grew at comparable rates and only occasionally were larvae observed to penetrate the cecal mucosa, a true tissue phase rarely occurs in the life cycle of H. gallinarum. With numbers and growth rate of lumen larvae as criteria of host susceptibility, 21-day-old chickens were the most susceptible to H. gallinarum.
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