W. M. Reid

Histomoniasis in gnotobiotic chickens and turkeys: biological aspects of the role of bacteria in the etiology.

Abstract The bacterial requirements for producing infectious enterohepatitis in bacteria-free chickens were different from those for the disease in bacteria-free turkeys. When both Escherichia coli and Clostridium perfringens were introduced with Histomonas meleagridis, mild atypical cecal lesions were produced in 3 of 28 chickens while typical liver and cecal lesions were both present in all of 25 turkeys. When single species of bacterial agents were used, the rate of infection was less. More lesions were produced with C. perfringens than with E. coli. The cecal bacterial flora of conventional chickens, when introduced into bacteria-free chickens, fulfilled the requirement of H. meleagridis for induction of infectious enterohepatitis. Attempts to define the specific bacteria involved in the etiology of the disease in chickens from among 48 different bacterial isolates and Candida albicans were unsuccessful, although very mild disease was produced inconsistently with various combinations of strains of E. coli with Pseudomonas aeruginosa and Streptococcus faecalis or with C. perfringens. Eimeria adenoides, a cecal coccidium, survived when introduced with Histomonos-infected Heterakis eggs into bacteria-free turkeys and supported the growth of some histomonads but not Heterakis gallinarium larvae. Eimeria adenoides did not simulate the role of bacteria in producing infectious enterohepatitis. Disease was produced and Heterakis larvae did survive in conventional turkeys receiving E. adenoides and infected Heterakis eggs. The essential contributing factor of bacteria in the pathogenesis of infectious enterohepatitis was determined to be neither a favorable pH nor an oxidation-reduction potential value within the intestine of the host. The oxidation-reduction potential of bacteria-free cecal contents was strongly positive, that of the conventional chicken with its indigenous microflora was strongly negative, while the potential of the contents of ceca with infectious enterohepatitis was intermediate. The pH of the bacteria-free cecum closely approximates that of the parasite-infected intestine, while the pH of the uninfected conventional cecum was somewhat lower. In the pathogenesis of infectious enterohepatitis, the role of bacteria apparently was more vital for the survival of Heterakis than Histomonas.

EFFECT OF THE ANTICOCCIDIAL ARPRINOCID ON PRODUCTION, SPORULATION, AND INFECTIVITY OF EIMERIA OOCYSTS

Medication of broilers with arprinocid [MK-302, 9-(2-chloro-6-fluorbenzyl adenine)] had 3 distinct effects on oocysts; (1) the number of oocysts produced was decreased, (2) fewer of the oocysts sporulated, and (3) those oocysts which did sporulate were less infective than those from unmedicated birds. The drug level necessary to prevent passage of oocysts depended on the species and strain of coccidia. To essentially eliminate oocyst production (less than 5% of controls) required medication with the following levels of arprinocid: 70 ppm with Eimeria maxima; 60 ppm with E. mivati, E. E. necatrix, and E. brunetti; and 50 ppm with E. tenella. With E. acervulina, oocysts were completely eliminated by 60 ppm of arprinocid with one field strain but were still numerous at 70 ppm with a second field strain. Oocysts recovered from birds on medication often failed to sporulate. No sporulation was seen at drug levels of 30 ppm or above with E. maxima and E. mivati. The level of arpinocid required to prevent sporulation with other species depended on the strain being studied, but varied from 30 ppm to 70 ppm. The oocysts of E. acervulina, E. mivati, E. tenella, and E. brunetti recovered from medicated birds that subsequently sporulated, were less infective when inoculated into susceptible birds, than oocysts from unmedicated birds. Oocysts from low medication level with E. necatrix (30 ppm) and E. maxima (10 ppm), once sporulated, were as infective as oocysts from unmedicated control birds, even though the numbers produced were less. No differences were detected in the time oocysts were produced between medicated and unmedicated birds infected with E. acervulina, E. maxima, E. brunetti, and E. tenella.

Effects of Eimeria acervulina on Intestinal pH in Conventional and Gnotobiotic Chickens

White Leghorn cockerels inoculated with sporulated oocysts of Eimeria acervulina had lower mean pH values in the upper, middle, and lower small intestine than did uninoculated birds. Conversely, the pH increased in the ceca while no decrease of pH was found in the gizzard. The magnitude of the pH decrease was not correlated with oocyst dose or severity of infection as measured by lesion scores. In gnotobiotic chicks, infection decreased pH in the duodenum from that in uninoculated chicks. The decrease was the same within any experiment, irrespective of microbial status of the birds (germ-free, Streptococcus fecalis inoculated as a monoisolate, or conventional bacterial flora), indicating that bacteria played no role in lowering the intestinal pH.

Pathogenicity of Eimeria brunetti in bacteria-free and conventional chickens.

In pathogenicity studies comparing bacteria-free and conventional chicks parasitized with Eimeria brunetti, mortality was 15/62 in bacteria-free birds as compared to 7/61 in chicks with conventional flora. Visual lesion scoring using a scale of 0 to 4, produced means of 2.6 in bacteria-free birds as compared to 2.4 in the conventional controls. There was no appreciable difference in weight gains, feed conversion or microscopic lesions between the two groups. Cecal lengths were shorter in bacteriafree chickens than in conventional controls. This difference was probably due to a previously unreported effect of the gnotobiotic environment. Gnotobiotic uninfected cecal lengths were similar to those of gnotobiotic infected birds. The prepatent period was 135 hr in both infected groups. Moderate coagulative necrosis of the lower small intestine, rectum and ceca was observed in bacteria-free birds as well as those with normal flora. This study establishes the fact that the presence of secondary microbial invaders is not essential for E. brunetti either to establish itself, or to induce pathological change. Intestinal parasites differ widely in their relationships to the microbiological flora present in the host digestive tract. At one extreme, Histononas meleagridis requires the presence of specific microorganisms for the production of the pathology typical of the disease, as established by Franker and Doll (1964) and Bradley and Reid (1966). On the other hand the tapeworm, Raillietina cesticillus, presents an entirely different picture in which the parasite appears to be unaffected by the presence or absence of bacterial microflora (Reid and Botero, 1967). Study of such interrelationships is of special interest to diagnosticians who have frequently speculated on the role of secondary bacterial invaders in influencing pathogenicity. Clark et al. (1962) conducted gnotobiotic studies with E. tenella and observed that there was very little difference in the pathogenicity of the coccidium between gnotobiotic and conventional chicks. There was, however, a delay of 12 to 15 hr in the appearance of the second generation merozoites in the feces of gnotobiotic chicks. In a more recent study, Visco and Burns (1966, pers. commun.) reported no mortality in 41 gnotobiotic chicks infected with Eimeria tenella as compared to Received for publication 20 August 1968. * University of Georgia College of Agriculture Experiment Stations Journal Series Paper 340, College Station, Athens, Georgia 30601. t Department of Parasitology, Veterinary College, University of Agricultural Sciences, Hebbal, Bangalore, India. t Department of Poultry Science, Auburn University, Auburn, Alabama 36830. 77% mortality in the infected conventional controls. They concluded that a close relationship exists between the host flora and E. tenella in the production of the cecal coccidiosis syndrome. Thus the effects of the bacterial flora on pathogenicity of this species remains unresolved. In the present study Eimeria brunetti was compared for pathogenicity in bacteria-free and conventional chickens. The necrosis originally described by Levine (1942) for infection of E. brunetti has been attributed by some to secondary invaders. MATERIALS AND METHODS Pathogenicity studies comparing the effects of Eimeria brunetti infection in bacteria-free chicks to those in birds with conventional microbiological flora were conducted in 1 preliminary (#1) and 3 (#2, 3, and 4) controlled trials. Sixty-two bacteria-free White Leghorn chicks were hatched and reared in 8 plastic film isolators. They were inoculated orally at 7 days of age with moderately heavy dosages of surface sterilized oocysts of E. brunetti. The quantity of inoculum (estimated at 110,000, 120,000, and 150,000 per bird in the 2nd, 3rd, and 4th trials, respectively) was determined by previous assay on conventional birds of the same age and breed. Selection of the number of oocysts in the inoculum followed a comparison of the effects of a graded series of oocyst numbers on mortality and morbidity using young susceptible birds. Sixty-one chicks of the same breed and age with conventional bacterial flora reared under the same conditions in 8 other isolators were similarly parasitized. The methods of Bradley et al. (1967) were adopted for rearing bacteria-free chicks. The coccidial oocysts were surface sterilized with 0.5% peracetic acid as described by Doll et al. (1963). In the preliminary trial (#1), 6 bacteria-free chicks reared in one isolator were inoculated with

Transmission of histomoniasis with male Heterakis gallinarum (Nematoda)

Histomoniasis was transmitted to poults inoculated orally with recently harvested whole intact male worms in three trials (4/5, 5/6, 2/4), but not with female worms (0/5, 0/9, 0/3). When triturated male worms were given in two trials, the rate of transmission was reduced (3/5, 0/10). Triturated female worms given orally produced histomoniasis in 1/9 poults. These results suggest that further unknown steps may be involved in histomonad transmission by heterakid worms. This study was partially supported by NSF Grant GB-5227.

Coccidiosis Immunity following Early and Late Exposure to Marek's Disease

SUMMARY White Leghorn cockerels were inoculated with plasma from chickens infected with Mareks disease at 1 or 28 days of age. Chicks were immunized against Eimeria tenella or E. maxima by administering oocysts at 1, 7, 14, and 21 days of age. Immunity to coccidiosis was tested at 35 days of age by challenge with coccidial species to which they had been immunized. Immunity to each species was increased if exposure to Mareks disease was delayed until the chicks reached 28 days of age.

Efficacy evaluations of robenzidene for control of coccidiosis in chickens

Robenzidene proved to be a potent anticoccidial agent when tested against severe coccidial infections in chickens maintained in batteries and floor pens. In laboratory experiments the drug was tested against inoculations of Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix, and E. tenella oocysts. E. praecox and E. hagani were also included in a combined infection tested in floor-pen experiments. No mortality, little (if any) depression in growth rate, and few lesions occurred in chickens medicated with robenzidene. Unmedicated controls showed 86.7% mortality in one laboratory experiment and up to 9.5% in floor-pen experiments. In tests involving lesion scoring and oocyst counts before and after withdrawal of the drug from the feed, both coccidiostatic and coccidiocidal properties were demonstrated.

Anticoccodial protection and development of immunity to Turkey coccidiosis while using monensin

Monensin at 60, 80 or 100 ppm provided sufficient early protection against coccidiosis in floor-pens seeded with a mixture of Eimeria adenoeides, E. gallopavonis and E. meleagrimitis oocysts to prevent mortality due to coccidiosis and permit satisfactory weight gains. Unmedicated poults suffered 7% mortality and significantly (P</= 0.05) reduced weight gains compared with medicated birds. No mortality occurred after medication was withdrawn at 10 weeks of age. In poults fed monensin at concentrations of 60, 80 and 100 ppm immunity development was sufficient to prevent mortality and weight loss in challenged poults. Some delay in development of immunity in birds medicated at 80 or 100 ppm was indicated by comparing lesion scores with those of unmedicated poults. Greater immunity occurred in all floor-pen reared groups (medicated or unmedicated) when compared with battery-reared, susceptible controls of the same age. Development of immunity, measured by lesion scores, occurred less rapidly in the caecal regions than in the upper intestine suggesting that E. meleagrimitis (upper intestine) may be more immunogenic than the other two species which largely parasitise the caecum.

Effects of Eimeria brunetti on intestinal pH in conventional and gnotobiotic chickens.

White Leghorn cockerels, 2-4 weeks of age, inoculated with sporulated oocysts of Eimeria brunetti had a lower mean pH in the middle and lower small intestine than did uninoculated controls by 5-9 days post-inoculation. No effect of the infection on pH was seen in the upper intestine while pH in the gizzard and caeca was occasionally higher than in controls. Magnitude of pH. decrease did not correlate with increases in oocyst dosage and severity of infection as measured by lesion score. Studies with germ-free (GF) and gnotobiotic chicks showed that host bacterial flora was essential for a pH decrease in the middle and lower small intestine with E. brunetti infection. Inoculation of GF birds with Lactobacillus acidophilus or L. acidophilus plus Streptococcus faecalis, in addition to E. brunetti resulted in some decrease in pH.

Efficacy of Arprinocid against Coccidiosis of Broilers in Battery and Floor-Pen Trials

Medication of broilers with arprinocid [9-(2-chloro-6-fluorophenylmethyl)-9H-purine-6-amine] gave protection against the effects of coccidiosis in both battery and floor-pen trials. In battery trials, efficacy was tested on single-species inoculations of Eimeria acervulina, E. mivati, E. necatrix, E. maxima, E. Brunetti, and E. tenella. Two strains of each species recently recovered from the field, were tested separately. In floor-pen trials, all six species, both field and laboratory strains, were used as a mixed infection. In batteries, 60 and 70 ppm essentially eliminated coccidiosis-induced mortality and weight depression. Effects of 50 ppm on weight gain were variable. The effectiveness of different medication levels varied between strains within a species. In floor-pen trials, 40, 60, or 80 ppm was effective in controlling mortality and weight depression, and increasing feel-conversion ratios. All levels were significantly as effective as monensin in protecting against coccidiosis. With severe exposure to coccidia, 60 and 80 ppm gave significantly lower lesion socres than did 40 ppm of arprinocid or 120 ppm monensin.