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• W1969901919 abstract "Bacteria-free and conventional turkeys were inoculated with bacteriologically sterile heterakid eggs containing Histomonas meleagridis. Only 1 of 12 gnotobiotic turkeys showed any signs of histomoniasis, and none died of the disease. Of 12 conventional turkeys, 11 developed histomoniasis and died. Heterakis gallinae was not recovered from the bacteria-free turkeys. The results suggest that either the infectivity of Heterakis or the pathogenicity of Histomonas, or both, may be related to normal host flora. Proof that Histomonas meleagridis is the sole etiological agent of blackhead in turkeys is lacking. The presence of bacteria and other protozoa in liver and cecal lesions has suggested the possibility of synergism. Protozoa found with Histomonas in necroses have been grown in pure culture and inoculated into turkeys, but any resultant lesions were different from those caused by histomonad infection (Allen, 1941; Harrison et al., 1954). Many instances of bacteria in tissue lesions have been recorded. However, Harrison et al. (1954) have produced bacteria-free histomoniasis lesions in the liver. In vitro culture of Histomonas in sterile media was unsuccessful until the recent work of Lesser (1960, 1961). In nature, the histomonad enters the host tissue via the cecum. Ulceration of the cecal wall permits entry of bacteria and Histomonas into the liver via the portal system. Secondary foci are usually established in the liver. The need for a study of the histomonad-bacteria relationship in this pathogenesis has been apparent for some time. The bacteria-free turkey is an excellent tool for such a study. Natural infection is generally the result of ingestion Received for publication 23 November 1962. * This study was supported by the Office of Naval Research, the Army Surgeon General, the National Institutes of Health, and the University of Notre Dame. t From research work done by the junior author at the University of Notre Dame as a partial fulfillment of his Ph.D. dissertation. of Histomonas-infected eggs of the cecal worm Heterakis gallinae. The present study describes the pathogenicity of Histomonas in the absence of viable host flora. The conditions of natural infection are closely simulated. MATERIALS AND METHODS Fertile Beltsville White turkey eggs were immersed in an aqueous 2% detergent solution (Turbo-Jet Egg Cleaner, Yargar Poultry Company, Warsaw, Indiana) for 5 min, air-dried on a sterile cloth, and incubated without rinsing in a commercial type incubator. On the 27th day, viable embryonated eggs were introduced into a Reyniers isolator unit (Reyniers et al., 1949; Luckey et al., 1960) via a 7-min dip in a germicidal trap containing 2% aqueous mercuric chloride. The control turkeys were obtained from similarly treated eggs, but were allowed to hatch in the incubator and then transferred to starter batteries in a conventional environment. On hatching, the experimental poults were immediately moved to ?-inch mesh wire-screen pens in the isolator to prevent ingestion of eggshell debris. This debris was removed from the isolator via a sterile lock and its bacteriological status determined in fluid thioglycollate medium incubated at 25, 37, and 55 C. If any growth was observed in this medium, the clutch was considered contaminated and not included in the data. Both groups were maintained on an autoclaved diet designated L-289F (Gordon, Doll, and Wostmann, 1958). Diet and sterile water were fed ad lib. An inoculum of bacteria-free Heterakis eggs was prepared in the manner described by Doll (1963). A sterile ampule containing the desired amount of inoculum was introduced into the isolator. Ten days after hatching, each bird received approximately 1,000 heterakid eggs by direct intubation into the proventriculus. The ampule with the 411 This content downloaded from 207.46.13.114 on Sun, 28 Aug 2016 06:18:25 UTC All use subject to http://about.jstor.org/terms 412 THE JOURNAL OF PARASITOLOGY Vol. 49, No. 3 TABLE I. Results of oral inoculation of bacteria-free and conventional turkeys with bacteriologically sterile heterakid eggs (1,000 per bird) containing H. meleagridis. Birds were inoculated 10 days after hatching. Bacteria-free turkeys Conventional turkeys No. of days to Pathology1 No. of days to Pathology1 Bird death (d) or Bird death (d) or number autopsy (a) Liver Ceca number autopsy (a) Liver Ceca 1 17(a) 0 0 1 14(d) 1 2 2 17(a) 1 0 2 14( ) 1 2 3 22 (a) 0 0 3 15(d) 1 2 4 22 (a) 0 0 4 15(d) 1 2 5 22(a) 0 0 5 15( ) 1 1 6 21(a) 0 0 6 16(d) 1 2 7 21 (a) 0 0 7 16( ) 1 2 8 21(a) 0 0 8 17(d) 2 2 9 34 (a) 0 0 9 18( ) 2 2 10 34(a) 0 0 10 20(d) 1 2 11 41 (a) 0 0 11 21 (d) 2 2 12 41 (a) 0 0 12 21(a) 0 0 1 0 = no lesions; Liver: 1 = few small necrotic foci; 2 = abundant large foci (7 mm or more in diameter). Ceca: 1 = ulceration and core restricted to proximal end; 2 = ulceration and core extending length of organ. remaining eggs was removed from the isolator, and the control group received a similar portion of the inoculum. Experimental poults were killed at intervals of from 17 to 41 days postinoculation. Autopsy was performed in a conventional laboratory environment at a maximum of 30 min after removal from the isolator. Gross lesions of the ceca and liver were recorded. The entire gut and cecum were examined for the presence of H. gallinae larvae. The intestinal tissue was trypsinized in 0.8% buffered solution at 40 C. After 36 hr this digest was centrifuged and examined for heterakid larvae and eggs. Portions of the ceca and liver were removed at autopsy and fixed in 10% neutral formalin. Dehydration, clearing, and embedding were accomplished in the usual way. Sections were stained with Mayer's hemalum and counterstained with aqueous eosin. Conventional birds were examined by a similar procedure at necropsy. The bacteriological status of the experimental group was determined at weekly intervals during the preinoculation and postinoculation period and at the conclusion of each experiment. Checks for bacterial sterility followed the technique described by Wagner (1959). In addition to this, portions of the liver from each turkey in the bacteria-free group were excised under sterile conditions, macerated, and tested for bacterial sterility." @default.
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• W1969901919 title "Experimental Histomoniasis in Gnotobiotic Turkeys. I. Infection and Histopathology of the Bacteria-Free Host" @default.
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