Important parasites in poultry production systems.

Poultry now accounts for 30% of all meat consumed. Parasites are a problem where ever poultry are raised, whether in large commercial operations or in small back-yard flocks, and economic losses can be significant. This paper will briefly review the major protozoan, helminth, and arthropod species in poultry including pathogenesis. Other topics will include the importance of the interaction of other diseases and parasites, and control of the infection by chemotherapy, management, and immunity.

Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences.

Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.

In vivo role of tumor necrosis-like factor in Eimeria tenella infection.

The effect of tumor necrosis-like factor (TNLF) on the pathogenesis of coccidiosis was investigated. Injection of crude chicken TNLF enhanced the weight loss caused by Eimeria tenella infection. Rabbit polyclonal antibody against recombinant human tumor necrosis factor-alpha (rhTNF) partially restored E. tenella-induced weight loss in SC chickens, but not in TK chickens. However, injection of chickens with chicken TNLF, rhTNF, and rabbit serum against rhTNF had no significant effect on cecal lesions. Both SC and TK chickens produced circulating TNLF following primary, but not secondary infection, and SC chickens showed higher level of TNLF production than TK chickens. Peripheral blood leukocyte-derived macrophages from SC and TK chickens produced a significant amount of TNLF compared to the preinfection condition when cocultured with sporozoites. In general, macrophages from SC chickens produced higher levels of TNLF than those from TK chickens. No significant difference was observed between primary and secondary infection. These results suggest that the excessive TNF production may be involved in weight loss caused by E. tenella infection in SC chickens.

Chicken tumor necrosis-like factor. I. In vitro production by macrophages stimulated with Eimeria tenella or bacterial lipopolysaccharide.

HD11, a transformed avian macrophage cell line, and chicken peripheral blood leukocyte-derived macrophages (PBL-M phi) were stimulated with bacterial endotoxin lipopolysaccharide (LPS) or Eimeria tenella sporozoites and merozoites. The specific cytotoxicities of the culture supernatants against different target cell lines were measured, and the kinetics of tumor necrosis-like factor (TNF) production by HD11 and PBL-M phi were also measured. The results showed that HD11 and PBL-M phi secreted a TNF-like factor when stimulated with Eimeria parasites or LPS. A time- and dose-dependent TNF-like factors production by PBL-M phi was observed poststimulation with Eimeria parasites. Chicken TNF-like factor preferentially kills CHCC OU-2 cells, a fibroblast cell line of chicken origin, when compared to LM cells, a murine cell line used for mammalian TNF. This study indicates that chicken M phi produce a significant level of TNF-like factor following coccidial infection.

Experimental toxoplasmosis in chukar partridges(Alectoris graeca).

Thirty battery-hatched chukar partridges (Alectorts graeca) were inoculated orally with oocysts of the ME 49 or the GT-1 strain of Toxoplasma gondii. All six chukars given 10,000 GT-1 strain oocysts died or were euthanized between postinoculation day (p.i.d.) 3 and 6. Fifteen of 24 chukars given 10,000, 1000, 100 or 10 ME 49 strain oocysts died or were euthanized between p.i.d. 6 and 14. Nine chukars that were not ill by p.i.d. 14 remained clinically normal until euthanized in good health p.i.d. 47 and 67; T. gondii was found by bioassay in mice inoculated with tissues of these nine chukars. From the tissues of five chukars bioassayed individually in mice, T. gondii was isolated from brains of four of four tested, and from the hearts and skeletal muscles of five, and livers of three of five chukars tested. Major lesions in chukars that died or those euthanized when ill were enteritis, splenic necrosis, myocarditis and encephalitis. Myocarditis and encephalitis persisted in chukars examined p.i.d. 47, 53 and 67. All chukars examined p.i.d. 10 developed anti-T. gondii antibodies. Anti-T. gondii antibodies detected in the modified agglutination test were higher than those in latex and haemagglutination tests. The Sabin-Feld-man dye test did not detect T. gondii antibodies in sera of chukars. The ME 49 strain of T.gondii was more pathogenic to chukars weighing >/= 300 g than to the 25 g Swiss Webster mice.

Experimental toxoplasmosis in Japanese quail.

Twenty-four 5-month-old battery-hatched Japanese quail were inoculated orally with 10(5) (ME 49 strain, group A, 6 birds), 10(3) (ME 49 strain, group B, 6 birds), 10(5) (GT-1 strain, group C, 6 birds), and 10(3) (GT-1 strain, group D, 6 birds) Toxoplasma gondii oocysts. All birds in group C died or were euthanized within 8 days after inoculation (DAI). Five of the 6 birds in group D died or were euthanized 8, 9, 15, 19, and 23 DAI. One of the 6 quail in group A died 9 DAI, and 1 of the 6 birds in group D died 16 DAI. The 11 quail (1 from group D and 10 from groups A and B) were euthanized 63 DAI; T. gondii was isolated by bioassays in mice from the brains of 10, hearts of 10, and skeletal muscles of all 11 quail. Quail that survived marked small intestinal and splenic toxoplasmosis lived long enough to develop severe protozoal pneumonia, myocarditis, or meningoencephalitis. The quail that survived only to be examined at 63 DAI had moderate multifocal nonpurulent encephalitis and myositis and had a hypertrophic spleen that contained hemosiderin-laden macrophages. Toxoplasma gondii antibodies were found in the sera of all quail examined 63 DAI. Antibody titers to T. gondii in the modified agglutination test were higher than in the indirect hemagglutination and latex agglutination tests. Antibodies were not detected in quail sera examined by the Sabin-Feldman dye test.

Experimental toxoplasmosis in pheasants (Phasianus colchicus).

Sixteen 6-mo-old battery-reared ring-necked pheasants (Phasianus colchicus) were inoculated orally with 10(5) (group A, ME 49 strain, five birds), 10(4) (group B, ME 49 strain, six birds) and 10(4) (group C, GT-1 strain, five birds) Toxoplasma gondii oocysts. The pheasants in groups A and B remained clinically normal. One of the pheasants in group C died 19 days after inoculation (DAI); T. gondii was found in histological sections of brain and heart and encephalitis, myocarditis and enteritis were the main lesions. Toxoplasma gondii was isolated by bioassays from pooled tissues of five of six pheasants in group B killed 36 DAI. Toxoplasma gondii was isolated from the brains, hearts and skeletal muscles of each of the four pheasants inoculated with the GT-1 strain (group C), and from the brains of four, hearts of three and skeletal muscles of four of five pheasants inoculated with the ME 49 strain (group A). All pheasants developed high (1: 1,600-1:25,600) antibody titers to T. gondii in the modified agglutination test (MAT) 36 to 68 DAI. Antibody titers detected with the MAT were higher than those detected in the indirect hemagglutination and latex agglutination tests. Antibodies were not detected in 1:4 dilution of pheasant sera with the Sabin-Feldman dye test.

Experimental toxoplasmosis in bobwhite quail (Colinus virginianus).

Twenty-four 3-5-mo-old battery-hatched bobwhite quail were inoculated orally with 10(5) (ME 49 strain, group A, 6 birds), 10(4) (ME 49 strain, group B, 6 birds), 10(5) (GT-1 strain, group C, 6 birds), and 10(4) (GT-1 strain, group D, 6 birds) Toxoplasma gondii oocysts. One quail in group B died 18 days after inoculation (DAI) due to undetermined etiology. Two quail from group C and 1 quail from group D given GT-1 oocysts died of acute toxoplasmosis 6, 7, and 8 DAI, respectively; numerous T. gondii tachyzoites were found in lesions in visceral tissues. The surviving quail in group A and B were killed 106 DAI and those in groups C and D were killed 60 DAI. Toxoplasma gondii was isolated from pooled tissues of 4 of 5 quail in group B. Brains, hearts, and skeletal muscles from quail in groups A, C, and D were bioassayed individually in mice; T. gondii was isolated from the brains of all 6, hearts of 3, and skeletal muscles of 2 of the 6 quail in group A; from the brains of all 4, hearts of 1, and skeletal muscles of 2 of the 4 quail in group C; and from the brains of all 5, hearts of 1, and skeletal muscles of 3 of the 5 quail in group D. Toxoplasma gondii antibodies were found in sera of all 20 quail killed 60 or 106 DAI. Antibody titers detected in the modified agglutination test using whole tachyzoites were higher than in latex agglutination and indirect hemagglutination tests that used soluble antigens. Antibodies were not detected in a 1:4 dilution of 19 of 20 quail sera in the Sabin-Feldman dye test.

Experimental toxoplasmosis in turkeys.

Fourteen 2-3-wk-old turkeys were inoculated orally with 10(5) or 10(4) infective oocysts of the ME 49 strain of Toxoplasma gondii. Of the 8 turkeys given 10(5) oocysts in experiment 1, 3 died or were killed 12 or 14 days after inoculation (DAI) because of respiratory distress associated with a concomitant Aspergillus-like fungus infection. The remaining 5 turkeys remained normal and were killed 62 DAI. Toxoplasma gondii was isolated in mice from the heart of all 5, from the breast muscles of 2, leg muscles of 3, and from the brains and livers of none of the turkeys. All 6 turkeys fed 10(4) oocysts in experiment 2 remained clinically normal until necropsy on 41 DAI; T. gondii was isolated from pooled tissues from each turkey. All 14 turkeys developed high antibody titers to T. gondii in the modified agglutination test (MAT) using formalinized tachyzoites. The enzyme-linked immunosorbent assay (ELISA) was as sensitive as MAT for detecting T. gondii antibodies in turkey sera. The latex agglutination and indirect hemagglutination tests were less sensitive than the MAT and ELISA. No dye-test-measurable antibodies were found in sera of any turkey.

Serologic and parasitologic responses of domestic chickens after oral inoculation with Toxoplasma gondii oocysts.

Four-week-old chickens were inoculated orally with 1,000 or 100,000 oocysts of the ME-49 or GT-1 strain of Toxoplasma gondii, and their antibody responses were measured, using the direct modified agglutination test, latex agglutination test, indirect hemagglutination test, ELISA, and the Sabin-Feldman dye test. Antibodies against T gondii were detected by use of the modified agglutination test and ELISA within 2 weeks of oocyst inoculation, and antibodies persisted until termination of the study by postinoculation day 68. The latex agglutination test was insensitive in detecting T gondii antibodies, and antibodies were not detected by use of the dye and indirect hemagglutination tests. Of tissues bioassayed in mice for tissue cysts by pepsin digestion of individual organs of chickens on postinoculation day 68, tissue cysts were found in the brain of all 5, heart of 3, and leg muscles of 2, but not in the liver and breast muscles. None of the birds developed clinical toxoplasmosis.

Effect of amprolium on production, sporulation, and infectivity of Eimeria oocysts.

Amprolium reduced the number of oocysts shed by Eimeria acervulina, E. maxima, E. necatrix, and a mixture of susceptible strains of E. tenella. Sporulation of oocysts from mediated chickens was reduced compared with that of oocysts from unmedicated chickens. Sporulation was reduced by levels of 0.0250% amprolium for E. acervulina and by levels of 0.0060% for E. maxima and the susceptible E. tenella. Not enough oocysts were recovered to measure sporulation of E. necatrix. Sporulation reduction was not affected by the method of administration of amprolium (feed or water), except with E. acervulina, for which fewer oocysts sporulated when 0.0120% amprolium was added in the drinking water than when 0.0125% amprolium was added to the feed. Conversely, amprolium medication had no effect on the sporulation of an amprolium-resistant E. tenella. When fed to unmedicated chickens, those oocysts from amprolium-medicated chickens that did sporulate were as infective as oocysts recovered from unmedicated chickens.

Characterization of the toxicity of the mycotoxins aflatoxin, ochratoxin, and T-2 toxin in game birds. III. Bobwhite and Japanese quail.

Bobwhite and Japanese quail were fed diets containing 1.25, 2.50, or 5.00 ppm aflatoxin; 1, 2, or 4 ppm ochratoxin A (OA); or 4, 8, or 16 ppm T-2 toxin. Aflatoxin induced mortality in bobwhites during the second and third week with 1.25 ppm (10%), 2.50 ppm (30%), and 5.00 ppm (40%), and during the same period with T-2 toxin at 8 ppm (20%) and 16 ppm (22.5%). Body weights of bobwhite quail were significantly decreased by the two higher levels of aflatoxin by 2 weeks of age, and by the two higher levels of T-2 toxin by 1 week of age. In Japanese quail, only the highest level of aflatoxin and T-2 toxin reduced body weight (by 3 weeks and by 1 week of age, respectively), and even then to a much lesser extent than in bobwhites (less than 10%). Aflatoxin did not affect feed-conversion ratio (FCR) in bobwhite quail, but the two higher levels of T-2 toxin increased FCR. None of the toxins induced mortality or increased the FCR in Japanese quail. Aflatoxin increased liver weight in both bobwhite and Japanese quail. OA increased kidney weight in 3-week-old Japanese quail but had no effect on the kidney weight of bobwhite quail. Mouth lesions were progressively more severe in bobwhite quail fed increasing levels of T-2 toxin, but lesions were far less severe in Japanese quail.

Characterization of the toxicity of the mycotoxins aflatoxin, ochratoxin, and T-2 toxin in game birds. II. Ringneck pheasant.

Ringneck pheasants were fed diets containing 1.25, 2.5, or 5 ppm aflatoxin; 1, 2, or 4 ppm ochratoxin A (OA); or 4, 8, or 16 ppm T-2 toxin. Severe toxin-induced mortality was seen during the first to third weeks with 2.50 and 5.00 ppm aflatoxin (92.5% and 97.5%, respectively), compared with the mortality in control pheasants fed no toxin (0%). Slight mortality (less than or equal to 5%) was seen with OA and T-2 toxin. Body weights were significantly decreased by the lowest level (1.25 ppm) of aflatoxin by 2 weeks of age, by the two highest levels of aflatoxin by 1 week of age, and by 16 ppm T-2 toxin by 1 week of age. The feed-conversion ratio was increased by 2.50 and 5.00 ppm aflatoxin compared with the feed-conversion ratio in controls, although high mortality may have influenced the results. Aflatoxin had no effect on liver weight, but OA increased kidney weight in 3-week-old pheasants. Mouth lesions were seen in some of the pheasants fed T-2 toxin.

Altered metabolism of carotenoids during pale-bird syndrome in chickens infected with Eimeria acervulina.

The progression of changes in carotenoid metabolism during pale-bird syndrome caused by a coccidial infection was investigated. Male broiler chickens 15 days of age on a yellow corn and soybean meal-based diet were infected with Eimeria acervulina oocysts and their serum, liver, and toe webs were sampled at 0, 4, 6, and 10 days postinfection for HPLC analysis of carotenoids. At 4 days postinfection a drastic reduction (71%) in serum lutein, the main body carotenoid, and smaller reductions in liver (58%) and toe webs (38%) occurred. Derivative forms of lutein, mainly esters, continued to be lost from tissues for 10 days postinfection. These carotenoids were apparently lost via the intestinal tract because birds placed on a white corn and soybean meal-based diet at time of infection had lutein in their jejunal contents even at 7 days postinfection. The loss of carotenoids from the body was accompanied by a decreasing ability to absorb canthaxanthin, a red carotenoid, from the intestinal contents. The absorption of canthaxanthin measured at 0, 3, 4, 5, 6, and 7 days reached its low point of 1% of preinfection ability on Day 5 before a slow recovery commenced. Thus, the pale-bird syndrome caused by E. acervulina appeared to be the result of a loss of previously absorbed carotenoids coupled with drastic malabsorption of dietary carotenoids.

Supplemental use of liquid amprolium in skip-a-day feeding of replacement pullets.

The addition of liquid amprolium to the drinking water on days when medicated (amprolium) ration was not fed in a restricted feeding (skip-a-day) program improved protection against a primary exposure to Eimeria acervulina and Eimeria tenella, yet still allowed for the development of protective immunity to subsequent challenge. With E. tenella, the best protection, as measured by reduction of lesion score, was provided by amprolium given in the drinking water on alternate days to feed medication when compared with the use of amprolium only in the feed or liquid amprolium at less frequent intervals (every second or third nonfeeding day). With Eimeria maxima, amprolium in the feed did not significantly lower lesion score compared with the score in unmedicated pullets; however, the further addition of amprolium to the drinking water did. When pullets were reared in floor pens previously seeded with coccidia, amprolium medication in the feed alone reduced the E. tenella-induced mortality rate from 28 to 8%. The addition of amprolium in the drinking water on nonfeeding days eliminated all deaths. Floor-reared pullets were caged after 3 wk and challenged 1 wk later with the same species of coccidial oocysts used to immunize on the floor. Coccidial lesion scores following challenge were eliminated or markedly lower than in pen-reared (unimmunized) pullets similarly challenged. This indicated that protective immunity developed despite the use of amprolium in the drinking water.

Prevention of coccidiosis in the chukar partridge (Alectoris chukar) by medication with sulfadimethoxine and ormetoprim (Rofenaid).

Rofenaid (a 5:3 mixture of sulfadimethoxine and ormetoprim) was effective in preventing coccidiosis of chukar partridges. Levels of .0100 or .0125% sulfadimethoxine, with the corresponding level of ormetoprim in the ration, gave the best results. These levels markedly reduced mortality in severe infections (over 75% mortality in unmedicated chukars) and eliminated mortality in medicated groups when infections produced 52% mortality or less in unmedicated controls. In the latter infections, Rofenaid also protected against the depression in weight gain seen 6 days postinoculation in unmedicated chukars. Studies in uninoculated chukars showed that Rofenaid has a wide safety margin and did not produce adverse effects even at levels of .0300% sulfadimethoxine. Rofenaid should, therefore, be an effective medication for the prevention of coccidiosis in chukars in the field. Data from the present studies are being submitted to the IR-4 Program of the Food and Drug Administration for consideration of approval for the use of Rofenaid in chukar partridges.

Characterization of the toxicity of the mycotoxins aflatoxin, ochratoxin, and T-2 toxin in game birds. I. Chukar partridge.

Chukar partridges were fed diets containing 1.25, 2.5, or 5 ppm aflatoxin; 1, 2, or 4 ppm ochratoxin A (OA); or 4, 8, or 16 ppm T-2 toxin. Toxin-induced mortality was seen during the third week with 4 ppm OA (12.5%) and 16 ppm T-2 toxin (15%), compared with the mortality in control chukars fed no toxin (2.5%). Body weights were significantly decreased by the highest level of aflatoxin at 3 weeks of age, by the highest level of OA by 2 weeks of age, and by 8 and 16 ppm T-2 toxin by 1 week of age. Aflatoxin did not affect liver weight and OA did not increase kidney weight in 3-week-old chukars. There was a slight decrease in kidney weight in chukars fed 4 ppm OA; however, the decrease was related to the decrease in body weight produced by the toxin. Mouth lesions were seen at all levels of T-2 toxin fed.

Genetically engineered antigen confers partial protection against avian coccidial parasites.

A fusion protein of beta-galactosidase and Eimeria tenella produced in a recombinant Escherichia coli strain was injected into chickens and elicited partial protection against an oral challenge with Eim. tenella parasites. The fusion protein contained a 31 kilodalton (kD) coccidial antigen designated as 5401. The DNA sequencing of the 5401 antigen-coding sequence revealed that this protein segment was highly negatively charged and strongly hydrophilic, and contained an amino-acid sequence repeated five times. A dose-titration study showed that immunizing chickens with a single subcutaneous injection of the 5401 antigen at 1,200 to 4,800 nanograms (ng)/bird in Freund's complete adjuvant decreased lesion scores, mortality, and feed conversions compared to unimmunized, challenged controls. Using the 1,200 and 2,400 ng/bird of the 5401 antigen, group weight gains were higher than for the unimmunized, challenged birds. In three other trials using the 5401 antigen at 2,400 ng/bird with light, medium, and heavy coccidial infections, significant protection was evidenced by reduced lesion scores, increased individual weight gains, or both. In addition, feed conversions were reduced when compared with unimmunized controls or birds immunized with a noncoccidial protein E. coli extract. Western blot analysis of sporozoite preparations with serum from 5401-immunized birds labeled two antigenic bands of 66 and less than 200 kD. These results indicate that the coccidial proteins produced in E. coli are potentially effective immunogens for protecting chickens against avian coccidiosis.

In vitro and in vivo characterization of avian reoviruses. II. Clinical evaluation of chickens infected with two avian reovirus pathotypes.

The effect of two avian reovirus isolates (2408 and 1733) on digestion and nutrient metabolism in infected chickens was assessed by an in vitro absorption assay and clinical blood chemistry analysis. Birds of various ages were inoculated orally and intratracheally with reovirus and sampled periodically for the respective assays. Transitory malabsorption was observed in the duodenum of birds infected with reovirus 2408. Conversely, increased absorption was detected in the ileum of these same birds. Clinical blood chemistry analyses of birds infected with both isolates revealed that severely affected birds had abnormally elevated plasma total protein, plasma albumin, and calcium levels. Decreases were found in percent bone ash and, due to abnormally high globulin levels, in albumin:globulin (A:G) ratios. A significant (P less than 0.05) correlation between body weights and total protein, albumin, A:G ratio, and bone ash was found in infected birds. The most pronounced metabolic and physiologic changes occurred in the severely affected birds, and, in general, pathogenicity of the isolates was reflected by the degree of metabolic change.

Eimeria acervulina and Eimeria tenella in 15.B-congenic White Leghorns.

Six Ea-B-congenic lines of chickens were used to study the role of the B histocompatibility complex in susceptibility and immunity to Eimeria tenella and E. acervulina. The B haplotypes were 2, 2, 5, 12, 13, or 19. Parental line 15I5 with the B15 haplotype (15I5-15) and a commercial White Leghorn (CWL) line were also used. The B-congenic lines were generally less susceptible to E. tenella than the CWL line based on weight gain, cecal lesion score, plasma pigment, and packed cell volume. Within the congenic lines, the 15I5-15 line was more resistant to E. tenella than the 15.6-2 and 15.7-2 lines. Using a level of infection of E. acervulina resulting in moderate disease, the 15.7-2 line was more susceptible than all other lines based on weight gain, and the 15.6-2 and 15.7-2 lines had greater intestinal lesion scores than the 15.15I-5 or 15I5-15 lines. The CWL line was most susceptible based on lesion score. Thus 15I5-15 chickens were more resistant to primary infection by several criteria than the 15.7-2 or 15.6-2 chickens infected with either coccidial species. A single immunization with 100 oocysts of E. tenella produced less immunity in the congenic lines than in the CWL, whereas four immunizations resulted in immunity in all lines except 15.15I-5. Immunization with E. acervulina produced good immunity to a challenge infection in all lines. These results suggest that the B complex has a detectable influence on resistance and susceptibility to avian coccidiosis, but it may play only a minor role in the development of immunity to a challenge infection.