Experimental Cryptosporidiosis in Broiler Chickens12

ENVIRONMENT AND HEALTH Experimental Cryptosporidiosis in Broiler Chickens 1,2 BYRON L. BLAGBURN3 and DAVID S. LINDSAY Departments of Pathology and Parasitology, College of Veterinary Medicine, and Animal Health Research, Alabama Agricultural Experiment Station JOSEPH J. GIAMBRONE Department of Poultry Science CHRISTINE A. SUNDERMANN Department of Zoology and Wildlife, Auburn University, Alabama 36849-3501 FREDERIC J. HOERR Alabama Department of Agriculture and Industries, C. S. Roberts Veterinary Diagnostic Laboratory, Auburn, Alabama 36831-2209 (Received for publication June 16, 1986) ABSTRACT Seven-day-old conventional broilers were inoculated either orally or intratracheally (IT) with 2.5 x 105, 5.0 x 105, or 2.0 x 106 oocysts of Cryptosporidium baileyi (32 birds for each dosage level per group; 192 birds total). Thirty-two birds served as unninoculated controls. Mean weekly weight gain and feed conversion were determined during a 5-week period. Carcass pigment was graded using a Roche Color Fan®. Fecal oocysts were calculated from random cage samples 6, 8, 11, 13, 15, 18, 20, 22, and 25 days after inoculation (DAI). Effects of C. baileyi on immune responses were examined for Newcastle disease virus-hemagglutination inhibition (NDV-HI) antibody, infectious bursal disease virus-enzyme-linked immunosorbent assay (IBDV-ELISA) antibody titers and delayed hypersensitivity (DH) in half of the birds in each group. Disease or death from cryptosporidiosis did not result from oral inoculation of C. baileyi. Signs of respiratory disease, consisting of rales, sneezing, and dyspnea were observed in all IT-inoculated birds 7 to 21 DAI. Seven deaths occurred in the IT-inoculated groups 14 to 21 DAI. At necropsy, lung parenchyma was gray, firm, and wet in the ventral region. Air sacs contained a foamy, white to gray, mucoid fluid. Histologic lesions in the air sacs and bronchi were epithelial hyperplasia, discharge of mucocellular exudate to the mucosal surface, thickening of the mucosa by cellular infiltrates, loss of cilia, and dilation of mucous glands. Weight gains for IT-inoculated birds were lower (P<.05) than controls from 14 to 21 DAI, although weight gains for the 5-week period were not significantly different. Pigmentation scores for birds given C. baileyi orally at medium and high levels and for all IT-inoculated birds were significantly lower than controls. Oocyst output was similar regardless of route or level of inoculation. No significant differences were noted in IBDV-ELISA titers between control and C. baileyi-infected chicks; however, significantly increased NDV-HI titers and decreased DH indices were observed in chicks that received C. baileyi oocysts IT at 5.0 x 105 and 2.0 x 106. (Key words: cryptosporidiosis, Cryptosporidium baileyi, chickens, respiratory disease) 1987 Poultry Science 66:442-449 INTRODUCTION

Cryptosporidium spp. are coccidian parasites that inhabit the microvillous border of epithelial

'Publication Number 1843 College of Veterinary Medicine, Alabama Agricultural Experimentation Station Journal Number 5-861137. Supported by grants from the College of Veterinary Medicine (ALV-155) and the United States Department of Agriculture (85-CRSR-2-2606). 3 Reprint requests.

surfaces of vertebrates (Anderson, 1982). Their roles as disease agents in naturally occurring and experimentally induced infections in mammals are well-documented (Tzipori, 1983; Current, 1985). Among avians, Cryptosporidium has been recovered from chickens, turkeys, quail, peacock chicks, black-throated finches, red-lored parrots, ring-necked pheasants, and domestic goose (Whittington and Wilson, 1985; Lindsay et al., 1986). Although the taxonomy of Cryptosporidium species parasitizing avians

442

CRYPTOSPORIDIOSIS IN BROILERS

is controversial, Currents a/. (1986) have given the name Cryptosporidium baileyi to the species found in the domestic chicken (Gallus gallus domesticus). This terminology will be used herein. Cryptosporidium baileyi parasitizes the cecum, bursa of Fabricius, cloaca, trachea, nasal cavities, infraorbital sinuses, larynx, and bronchi of broiler chickens, but clinical cryptosporidiosis occurs only when the respiratory tract is infected (Fletcher et al., 1975; Dhillion etal., 1981;Randall, 1982; Itakuraef al., 1984; Lindsay and Blagburn, 1986; Lindsay et al., 1986). Most of the cited reports represent natural outbreaks in which C. baileyi was present with other potential pathogens. The primary pathogenic potential of C. bailey remains unknown. The present study clarified the pathogenic role of C. baileyi in the absence of demonstrable bacteria and Mycoplasma in 7day-old conventional broilers and measured its effect on growth performance, feed conversion, carcass quality, and immune responses following inoculation of oocysts into the respiratory and enteric tracts. MATERIALS AND METHODS

Chickens. Equal numbers of male and female broilers were obtained commercially and raised to 7 days of age prior to initiation of the studies. Birds were kept in groups of 8 in wire-floored cages and were fed an Auburn University broiler starter ration and water ad libitum. Preparation and Administration of Cryptosporidium baileyi oocysts. Oocysts of C. baileyi (AU-B1 isolate), obtained by bursal scraping from naturally infected broilers, were propagated by passage in coccidia-free chickens. Preparation and calibration of inocula were identical to procedures described by Lindsay et al., 1986. Oral inoculations were performed by placing oocysts in approximately .5 ml of Hanks' balanced salt solution (HBSS) (Grand Island Biological, Grand Island, NY) in a sterile syringe and introducing the syringe in the crop. Intratracheal (IT) inoculation was performed by oocysts placed in .1 ml of HBSS in a syringe fitted with an 18-gauge animal feeding needle (Popper and Sons, New Hyde Park, NY). The needle was placed in the trachea and the contents deposited slowly to avoid regurgitation. Effects of Cryptosporidium baileyi Infections on Weight Gain, Feed Conversion, and Carcass Quality. Four replicates of eight 7-day-old chicks (32 chicks/dosage level) were inoculated

443

orally with 2.5 x 105 (low dose), 5.0 x 105 (medium dose), or 2.0 x 106 (high dose) oocysts (96 chicks for all oral dose titration studies). Birds were weighed and ranked by weight on the day prior to inoculation. Birds were allocated to groups so that mean group weights were approximately equal. The same numbers of oocysts were inoculated IT into the same number (96) of chicks. Birds given oocysts via different routes were maintained in separate rooms. Four replicates of 8 chicks each (32 total) served as controls. Procedures for handling were identical to experimental birds, except chicks were not given C. baileyi oocysts and were kept in a separate room. Chicks were monitored daily for signs of disease. Birds that died were subjected to postmortem examinations including evaluations of paraffin sections or mucosal smears of trachea, air sacs, lungs, bursa of Fabricius, and cloaca. Samples of trachea and air sacs were cultured on blood agar and in Frey's medium for bacteria and Mycoplasma. Feces were monitored for oocysts 3 to 5 days after inoculation (DAI) using a Sheather's coverslip flotation technique (Ivens et al., 1978). Oocysts per gram of feces were measured for feces randomly collected from the cage bottoms of each replicate on 6, 8, 11, 13, 15, 18, 20, 22, and 25 DAI with C. baileyi. Oocyst concentration was calculated from oocysts counted with the aid of a hemacytometer after addition of 5 g of feces to 95 ml tap water. Birds were weighed weekly beginning the day prior to inoculation until the termination of the experiments. Feed consumption was measured by weighing feed prior to placement in feeders and by feed weighbacks weekly. Feed conversion was estimated by feed consumed divided by body weight gain. Half the birds in each treatment and control replicate were killed by cervical dislocation 28 DAI. The remainder were terminated 35 DAI. Carcass pigmentation was measured by placing a Roche Color Fan® against the shank of each bird immediately prior to cervical dislocation. Scores are from 0 to 10 based on increasing pigmentation. Effects of Cryptosporidium baileyi Infections on Immune Responses. Four birds in each of four replicates per dosage were injected intramuscularly with .4 ml of Freund's complete adjuvant and .5 ml of an inactivated commercial vaccine containing Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) (Maine Biological laboratories, Water-

444

BLAGBURN ET AL.

ville, ME) in each of two sites over the breast muscle 7 days after inoculation with C. baileyi. Two weeks after receiving the injections, blood was obtained by cardiac puncture for determination of serum, NDV-hemagglutinin (HI) antibody and IBDV-enzyme-linked immunosorbent assay (ELISA) antibody (Giambrone et al., 1985a). Birds were also injected with .2 ml of avian oil tuberculin intradermally in the wattle skin to test for DH (Giambrone, etal., 1985a). Statistical Analyses. Data were analyzed by standard SAS analysis of variance with treatment differences determined by Duncan's (1955) multiple range test as modified by Kramer (1956). Statements of statistical significance are based on P<.05. RESULTS

Oocysts were detected in the feces of chicks in all infected groups 5 DAI with C. baileyi. Oocysts were not recovered from the feces of control chicks at any time during the study. Signs of disease were not observed after oral inoculation of oocysts. However, chicks inoculated IT displayed signs of respiratory disease beginning 7 DAI. Initial signs were mild and consisted of sneezing and increased breathing rates. Severe respiratory disease was first seen approximately 14 DAI. Affected birds were dyspneic and sneezing and extended their heads to facilitate breathing. Severely affected birds were recumbent on their sterna and reluctant to move. Seven deaths occurred among chicks given C. baileyi oocysts IT; four deaths in the low dose group and three in the high dose group. Postmortem examination of these birds revealed lungs that were gray, firm, and wet in the ventral region. All had severe airsacculitis (Figure 1) characterized by accumulation of copious amounts of foamy, white to gray, mucoid fluid in the thoracic and abdominal air sacs. The frothy exudate was also present in the trachea. Histologic lesions in the respiratory mucosa were characterized by epithelial hyperplasia, discharge of mucocellular exudate to the mucosal surface, progressive thickening of the mucosa by lymphocytic infiltrates, and loss of cilia (Figure 2). In heavy infected mucosa, mucous glands were dilated and the epithelium attenuated. Lesions strongly suggested loss of mucociliary function, and since exudate in the tertiary bronchi resembled that found in the proximal respiratory tract, bronchopneumonia was considered a sequela to changes in the

bronchial mucosa (Figure 3). Tertiary bronchi, atria, and air capillaries were filled with Cryptosporidia, heterophils, mucous, and fibrin. The air capillary interstices were expanded by histiocytes, small mononuclear cells, and fewer heterophils. Regions of air sacs lined with respiratory mucosa underwent identical changes as other respiratory mucosal surfaces (Figure 4). There was marked inflammatory thickening, surface exudate as in the tertiary bronchi, and focal ulceration. Other areas of air sacs and also mesentery were thickened by histiocytes, lymphocytes, and plasma cells. No lesions were found in the intestinal tracts. Escherichia coli was recovered from the trachea of one dead bird. Two deaths occurred among birds given C. baileyi orally: one in the low dose group and one in the high dose group. Necropsy did not reveal lesions in the bursa or cloaca, although numerous developmental stages of C. baileyi were seen in mucosal smears of these organs. No developmental stages of C. baileyi were found in smears of the trachea of either chick nor were pathogenic bacteria or Mycoplasma cultured from their tracheas or air sacs. Numbers of oocysts recovered from the feces of orally infected and IT-infected chicks differed significantly on Day 11. At that time, chicks in the oral high group passed greater numbers of oocysts than chicks in other groups (Figure 5). Significant differences in counts observed on Day 15 did not reflect effects of route or levels of inoculation, because only chicks in the oral medium and IT, low groups passed greater numbers of oocysts than chicks in other groups. The peak period of oocyst output was from 8 to 11 DAI. Thereafter, oocyst counts decreased markedly until Day 25, when numbers were undetectable in most groups (Figure 5). Results of growth performance are presented in Table 1. Although weight gains for surviving birds did not differ significantly over the 5-week period, significant differences in weight gains were observed during Week 3, the time when birds were suffering from the severe effects of respiratory cryptosporidiosis, and also the period when deaths due to respiratory disease were observed (Table 2). Feed conversions differed significantly, although no conclusions could be drawn from the results (Table 1). Mean Roche scores of birds receiving C. baileyi oocysts at the oral medium, oral high, and tracheal low, medium, and high doses were significantly lower than uninoculated controls (data not shown).

3

2

1

2.0ab

2.3b

2.2 b

19ab

2.0ab

308.7 a

307.0 a

290.1 a

310.4 a

2.1

304.6"

316.3

2.7C

2.4 e

2.7 C

27bc

3.0 b

3.5

86.6 b

95.9 b

11.4 a

1.3 a

4.4 a

7.1

a

5.4 a

NDV-HI titer

59.8 a

52.9 a

59.8 a

47.3 a

28.0 a

36.3 a

59.9 a

IBDV-E

Serology2

Delayed hypersensitivity index = Mean antigen injected skin thickness of sensitized birds minus mean of skin thickne

Arithmetic mean of Newcastle disease virus-hemagglutination inhibition antibody (NDV-HI) and infectious bursal di

Average in grams of five weekly means. Mean for Week 5 was for one-half the chicks.

' ' Within columns, values followed by different letters are significantly different (P<.05).

Intratracheal low (2.5 X 10 s ) Intratracheal medium (5.0 X 10 s ) Intratracheal high (2.0 X 1 0 ' )

Oral low (2.5 X 10 s ) Oral medium (5.0 X 10 s ) Oral high (2.0 X 10 6 )

a

3.6 a

ab

1.8a

314.1 a

Uninoculated

a

Roche fan

Feed conversion

Gain

Treatment

Weekly mean 1

TABLE 1. Effects of respiratory and enteric Cryptosporidium baileyi infections in br

446

BLAGBURN ET AL.

FIG. I. Abdominal airsacculitis in a broiler 14 days after intratracheal inoculation with Cryptosporidium baileyi oocysts. Air sacs are covered with a foamy, gray, fluid exudate (arrows). FIG. 2. Secondary bronchus from broiler 14 days after intratracheal inoculation with Cryptosporidium baileyi oocysts. Note the presence of exudate in the lumen, developmental stages of C. baileyi on the epithelial surface (arrows), and mucosal thickening due to epithelial hyperplasia and infiltration of inflammatory cells. x333. FIG. 3. Lung from broiler 14 days after intratracheal inoculation with C. baileyi oocysts. Bronchopneumonia is evident in the tertiary bronchi and air capillaries. x67. FIG. 4. Air sac from a broiler with Cryptospordium-'mduced airsacculitis. The air sac membrane is thickened by lymphocytes, plasma cells and histiocytes. Developmental stages of C. baileyi are on the surface of the respiratory epithelium (arrows). x333.

DISCUSSION

Although NDV-HI titers for chicks receiving C. baileyi intratracheally at the low dose were not significantly different from either the control or orally inoculated birds, their titers were larger than birds in these groups. Differences were statistically significant in NDV-HI titers for chicks receiving medium and high intratracheal doses of C. baileyi. The DH reactions were significantly reduced in chicks receiving medium and high oocysts numbers via the IT route.

Results corroborate field observations indicating that respiratory cryptosporidiosis can be a severe and fatal disease of broilers. Overt respiratory signs resulted from inoculation of oocysts into the respiratory tract, regardless of the inoculum dose. Clinical signs were similar to those reported for naturally occurring disease (Dhillion et al., 1981; Itakura, et al., 1984), but mortalities were lower in this study, Dhillon

2000 f ID

1800

ORAL

ORAL MEDIUM(5.0xl0 5 )

1600

ORAL HIGH(2.0xl0 6 )

U.

1400

o < or o - . 1200

£2 to

>o

o o z <

LOW(2.5*.\(f)

] INTRA TRACHEAL L0W(2.5xl0 5 ) INTRA TRACHEAL MEDIUM(5.0xl.05) INTRATRACHEAL HIGH(2.0xl0 6 )

1000 800 600 400

UJ

200 0

JL 0

INOCULATION

6

II DAYS

13 AFTER

15

18

20

22

25

INOCULATION

FIG. 5. Cryptosporidium baileyi oocyst excretion by broiler chicks inoculated orally or intratracheally at three inoculation doses. Line above bar is 1 standard deviation. Bars with asterisks represent groups with significantly greater (P<.05) oocyst outputs for that day.

CRYPTOSPORIDIOSIS IN BROILERS

447

I

* m « '*,

# •

• .:# e . J


, t* M

w

*

'*

•

,

m

.

»

'

f

,

.

Mi « '

4H|

^HMHHHHERBRBHBSB

3

• >

9

2-

BLAGBURN ET AL.

448

TABLE 2. Effects of respiratory and enteric Cryptosporidium baileyi infections on weight gain and feed conversion 14 through 21 days after inoculation Feed conversion2

Treatment

Gain1

Uninoculated

342.0ab

Oral low (2.5 X 10 s ) Oral medium (5.0 X 10 s ) Oral high (2.0 X 10 6 ) Intratracheal low (2.5 X 10 s ) Intratracheal medium (5.0 X 10 s ) Intratracheal high

1.8a

a

2.2a

352.3b

2.1a

369.2b

2.1a

264.1C

3.0b

239.9C

2.1a

268.4C

2.4a

313.6

(2.0 X 10 6 )

' '^Vithin columns figures followed by different letters are significantly different (P<.05). 1

Grams.

2

Feed consumed (g) -J- body weight gain (g).

et al., (1981) reported heavy losses that they suggest were due to natural respiratory cryptosporidiosis in a flock of 16,000 7-week-old broilers. However, an adenovirus was recovered from the naturally diseased birds. Adenoviruses are potentially copathogenic in the respiratory tract of chickens (Dhillon and Winterfield, 1984). Greater losses experienced in that study were probably the result of combined effects exerted by both virus and protozoan. Clinical disease did not result from oral inoculation of oocysts in the present study, nor were significant effects observed on weight gain or in feed consumption in orally inoculated chicks. This also agrees with previous reports of the presence of C. baileyi developmental stages in the bursa of Fabricius or cloaca (or both) of broiler chickens and the absence of clinical disease (Fletcher et al., 1975; Lindsay and Blagbura, 1986; Lindsay etal., 1986). Severe air sac disease described in this and in a previous report, which resulted in heavy carcass condemnation at processing (Dhillon et al., 1981), places C. baileyi among those agents to be considered in the respiratory disease complex. Lesions associated with induced respiratory cryptosporidiosis in this study were more severe than those reported for naturally occurring disease (Dhillon et al., 1981; Itakura et al., 1984). Lesions common in natural and experimental disease include cystic dilation of mucus glands, mucosal inflammatory infiltrates, and

generalized epithelial deciliation. The recovery of E. coli from only one bird suffering from cryptosporidiosis indicates that C. baileyi can be a primary pathogen in broiler chickens. Field reports made interpretation of the role of C baileyi in respiratory disease difficult because of the overt presence of other pathogens. Our performance studies indicated that acute respiratory cryptosporidiosis exerts a significant effect on weight gain. Birds that survive the acute phase (7 to 21 DAI) of respiratory disease recover, however, and do not differ measurably from uninfected or orally infected birds. It is interesting, however, that feed conversions were nonsignificantly better for convalescing birds than for controls during Week 4. Weight gains for Weeks 4 were also higher for two of the three groups of recovering birds than for controls, although statistical differences were not evident for either of these parameters (data not included). Such observations are also seen with chicks suffering from coccidiosis caused by Eimeria tenella and E. necatrix. Maximum depressions in weight gains occur from 5 to 7 DAI, the period of acute disease. Thereafter, rebound weight gain and feed conversion superior to uninfected controls are often observed in surviving birds (Waletzky, 1970). Cryptosporidium baileyi oocyst production in chickens is similar to oocyst production for Eimeria species (Kheysin, 1972). Peak production occurs on the 2nd to 4th day of patency

CRYPTOSPORIDIOSIS IN BROILERS

and gradually declines until oocysts are no longer present. Mortality and severe morbidity are usually seen near the period of peak oocyst output for Eimeria spp. (Blagburn and Todd, 1984). The same pattern occurred in this study after IT inoculation of C. baileyi oocysts. The dynamics of C. baileyi oocyst output in broilers and the association of peak oocyst output with morbidity and mortality is similar to coccidiosis caused by Eimeria sp. Reductions in cell-mediated immunity (CMI) based on DH index were statistically significant in chicks receiving medium and high levels of C. baileyi oocysts via the IT route. Because CMI is important in the modulation of Eimeriainduced coccidial infections in chickens (Giambrone et al., 1980), reduced CMI is likely to be significant in birds infected with C. baileyi. Significant differences were not observed in birds receiving C. baileyi orally at any of the levels or IT at the low dose. Attention must also be given to the Cryptosporidium-infecttd bird's ability to respond to other agents such as viruses and intracellular bacteria when present concurrently with C. baileyi. Because the thymus was not examined in any of the infected birds, it was not possible to correlate thymic lymphoid depletion with decreased CMI. Such correlations have been observed in previous studies (Giambrone etal., 1985b). This study confirms the significance of respiratory cryptosporidiosis in broilers. The primary disease potential of C. baileyi was proven in the absence of other demonstrable pathogens, and the negative effects of cryptosporidiosis on growth performance and carcass pigment were clearly shown, especially during the acute phase of the disease. Results of the present study corroborate our previous results whereby oral and intracloacal inoculation of C. baileyi oocysts resulted in development of Cryptosporidia in the bursa and cloaca without development in the respiratory tract (Lindsay and Blagburn, 1986; Lindsay et al., 1986). None of the birds in these studies that harbored Cryptosporidia in the cloaca and bursa alone developed clinical cryptosporidiosis. Thus, bursal and cloacal cryptosporidiosis in the absence of pulmonary infection or other pathogens appears insignificant.

ACKNOWLEDGMENTS

The authors would like to thank Rebecca H. Mysinger, James F. Davis, Joy Vaughan,

449

Natalie Purcell, and Ronald P. Clay for technical assistance. REFERENCES Anderson, B. C , 1982. Cryptosporidiosis: A review. J. Am. Vet. Med. Assoc. 180:1455-1457. Blagbum, B. L., and K. S. Todd, Jr., 1984. Pathological changes and immunity associated with experimental Eimeria vermiformis infections in Mus musculus. J. Protozool. 31:556-561. Current, W. L., 1985. Cryptosporidiosis. J. Am. Vet. Med. Assoc. 187:1334-1338. Current, W. L., S. J. Upton, andT. B. Haynes. 1986. The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens. J. Protozool. 33:289-296. Dhillon, A. A., H. L. Thacker, A. V. Dietzel, and R. W. Winterfield. 1981. Respiratory cryptosporidiosis in broiler chickens. Avian Dis. 25:747-751. Dhillon, A. S., andR. W. Winterfield. 1984. Pathogenicity of various adenovirus serotypes in the presence of Escherichia coli in chickens. Avian Dis. 28:147-153. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. Fletcher, O. J., J. F. Munnell, and R. K. Page, 1975. Cryptosporidiosis in the bursa of Fabricius of chickens. Avian Dis. 19:630-639. Giambrone, J. J., U. L. Diener, N. D. Davis, V. S. Davis, V. S. Panangala, and F. J. Hoerr, 1985a. Effect of purified aflatoxin on turkeys. Poultry Sci. 64:859-865. Giambrone, J. J., U. L. Diener, N. D. Davis, V. S. Panangala, and F. J. Hoerr, 1985b. Effects of purified aflatoxin in broiler chickens. Poultry Sci. 64:852-858. Giambrone, J. J., P. H. Klesius, and S. A. Edgar, 1980. Avian coccidiosis: Evidence for a cell-mediated immune response. Poultry Sci. 59:38^13. Itakura, C , M. Goryo, andT. Umemura, 1984. Cryptosporidia! infection in chickens. Avian Pathol. 13:487^199. Ivens, V. R., D. L. Mark, andN. D. Levine, 1978. Principal Parasites of Domestic Animals in the United States: Biological and Diagnostic Information. Spec. Publ. 52, Coll. Vet. Med. and Agric., Univ. 111., Urbana, IL. Kheysin, Y. M., 1972. Continuity of coccidian infection. In: Life Cycles of Coccidia of Domestic Animals. K. S. Todd, Jr., ed. Univ. Park Press, Baltimore, MD. Kramer, C. V., 1956. Extension of multiple range tests to group means and unequal numbers of replication. Biometrics 12:307-310. Lindsay, D. S., andB. L. Blagburn. 1986. Cryptosporidium infections in chickens produced by intra-cloacal inoculation of oocysts. J. Parasiol. 72:615-616. Lindsay, D. S., B. L. Blagburn, C. A. Sundermann, F. J. Hoerr, and J. A. Ernest, 1986. Experimental Cryptosporidium infections in chickens: Oocysts structure and tissue specificity. Am. J. Vet. Res. 47:876-879. Randall, C. J., 1982. Cryptosporidiosis of the bursa of Fabricius and trachea in broilers. Avian Pathol. 11:95102. Tzipori, S., 1983. Cryptosporidiosis in animals and humans. Microbiol Rev. 47:87-96. Waletsky, E., 1970. Laboratory anticoccidial evaluation trials: review of designs, variables, criteria, and predictive value for field use. Exp. Parasitol. 28:42-62. Whittington, R. J., and J. M. Wilson. 1985. Cryptosporidiosis of the respiratory tract in a pheasant. Aust. Vet. J. 62:284.