Epidemiological aspects of the use of live anticoccidial vaccines for chickens

This review address the epidemiology (epizootiology) of coccidiosis in commercial chickens with emphasis on the effects on the use of live vaccines. Surveys suggest that all seven valid species of chicken coccidia (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella) are ubiquitous. All species are pathogenic to various extents. New results are presented on the pathogeneicities of E. acervulina, E. mitis and E. Praecox. Unless ingested by chickens, oocysts in poultry-house litter may die after about 3 weeks. Oocyst sporulation may be better in drier, rather than wetter, litter. Whether sporulated or not, up to 20% of ingested oocysts may pass undamaged through a chicken's intestine. The excreted, sporulated oocysts can be immediately reingested to initiate an infection; the unsporulated oocysts can still sporulate after passing through the intestine. The seven species differ in their times of appearance in commercial flock; hence particular vaccines may be designed for rearing standard broilers for up to about 6 weeks or for breeding stock. Attenuated, precocious lines of Eimeria in vaccines have low reproductive potentials, thus avoiding crowding, developing optimally, and stimulating immune response with minimal tissue damage. Cross-immunity between Eimeria species is probably minimal. There is reciprocity between the immune status of chicken and their excretion of oocysts for each species, ensuring continual stimulation of immune responses in birds on litter. Paracox vaccine, comprising all seven Eimeria species, is shown here to stimulate immunity to each of them independently. Total oocyst accumulation in litter following Paracox vaccination at 1 week comprises a small peak of vaccinal oocysts at 2-4 weeks, then a higher peak of the local virulent population at 4-7 weeks, which rapidly wanes. The attenuated drug-sensitive vaccinal oocysts probably interbreed with the corresponding wild species, reducing both virulence and drug-resistance in the local population. Anticoccidial vaccines may not induce complete immunity in chickens with lowered immunocompetence due to stressors, including certain viral disease. Future development of live vaccines for standard broilers may be expected in the relatively short term. The useful lives of anticoccidial drugs might be extended by rotating them with live vaccines.     

T lymphocyte roles during Eimeria acervulina and Eimeria tenella infections

This study evaluated the effects of selective depletion of T lymphocytes on Eimeria infections in chickens. Cell depletions were initiated in day- or week-old Hyline SC strain chickens using intra-peritoneal injections of monoclonal antibodies to CD4, CD8, or T cell receptor (TCR) alpha/beta. Control chickens received injections of irrelevant monoclonal antibody or phosphate buffered saline (PBS). Following the establishment of cell depletion, chickens were infected orally with E. acervulina or E. tenella, 1 x 10(4) oocysts for primary infections and 2 x 10(5) oocysts for secondary infections. Chickens treated with anti CD4 monoclonal antibody produced significantly more oocysts than controls following primary E. tenella but not E. acervulina infections. Development of resistance to challenge infection was unaffected. These results suggest that CD4+ lymphocytes are important in controlling primary infection with E. tenella. Chickens treated with anti-CD8 or anti-TCR alpha/beta monoclonal antibodies produced significantly fewer oocysts than controls following primary infection but significantly more oocysts than controls following secondary infection with both E. tenella and E. acervulina. Additionally, anti-CD8 treatment abrogated resistance to challenge infection. CD8-depleted chickens may exhibit decreased oocyst production following primary infection due to a lack of CD8+ lymphocytes to serve as transporting cells for sporozoites. The abrogation of resistance to secondary infection in CD8- and TCR alpha/beta-depleted chickens suggests that these cells are necessary for the development of protective immunity to coccidia.     

The effects of shuttle programs upon the growth of broilers and the development of immunity to eimeria species

A floor-pen trial was conducted to investigate the effects of different shuttle programs upon the growth of broilers to 8 wk of age. Nicarbazin, halofuginone, and robenidine, when included in the starter feed for 3 wk, were effective in preventing lesions due to Eimeria. The effects of medication upon performance were apparent, medicated groups gaining more weight by 6 wk and having a lower feed conversion at 6, 7, and 8 wk than the unmedicated controls. There were no significant differences in body weight at 6, 7, or 8 wk or feed conversion at 6 or 7 wk among the medicated groups, whether medication was withdrawn for 7 or 14 days. A decrease in the number of small and medium oocysts in the litter was observed as the trial progressed. Few large oocysts (Eimeria maxima) were seen in the medicated groups. Numbers of oocysts did not increase following withdrawal of medication. Birds from all medicated groups were challenged at 6 wk with oocysts of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. Weight gains were similar to that of the unchallenged controls, indicating that they had acquired immunity to these species of Eimeria.     

Nucleic acid and protein synthesis in the body of chicks infested by various species of Eimeria

The infection of chicks with optimal immunogenic doses of oocysts of E. tenella and E. acervulina results in the activation of biosynthesis of proteins and nucleic acids in the Fabricius bursa, spleen, thymus and blind processes of the intestine. The infection causes the increase in the quantity and synthesis intensity of immunoglobulins G and M that suggests a stimulation of cellular and immunity factors in the ogranism of an immunized bird. Maximum changes in biosynthesis of compounds studied are reported four days after the infection that confirms high immunogenic properties of the second generation schizonts. A study of protein synthesis in limphoid organs of chicks infected only with E. tenella has yielded the same results. The infection with E. acervulina caused but negligible changes in the protein metabolism, i.e. coccidians of E. tenella possess more immunogenic properties.     

Prevention of Eimeria alabamensis coccidiosis by a long-acting baquiloprim/sulphadimidine bolus

Twelve calves aged 6-10 months, and 12 calves aged 10-16 months were turned out onto a permanent pasture known to have been contaminated with oocysts of Eimeria alabamensis during the previous year. Two days after turnout, six of the older calves and six of the younger were each treated with one bolus per 200 kg bodyweight containing 1.6 g baquiloprim and 14.4 g sulphadimidine. The other 12 calves were left untreated. The excretion of Eimeria oocysts, the faecal dry matter and the weight gain of treated and untreated calves within each age group were compared during the first 3 weeks on pasture to assess the efficacy of the bolus in preventing E. alabamensis coccidiosis. All the older of the untreated calves and four of the younger developed gruel-like to watery diarrhoea 4-7 days after turnout. The faecal consistency of the treated calves remained firm and they lost significantly less weight than the control calves during the first 13 days on pasture. The treated calves also excreted significantly fewer oocysts during the first 20 days of grazing; their oocyst excretion remained low during days 8-10 when all but one of the diarrhoeic control calves excreted more than 850,000 oocysts per gram faeces (OPG). Starting on days 12 to 14 the oocyst excretion of 8 of the treated calves increased to 20,000-65,000 OPG and of 2 calves to 210,000-240,000 OPG. There was no difference in oocyst output between treated and untreated calves from the fourth week of grazing and no difference in weight gain among the younger calves. In the older calves there was a tendency for the untreated calves to gain more weight than treated calves.     

Recent management of pituitary adenomas

Eight 2-day-old SPF chickens were each inoculated orally with a single dose of 5 x 10(5) oocysts of Cryptosporidium baileyi, and immunoglobulin G (IgG) antibody responses were chronologically measured by indirect immunofluorescent antibody (IFA) assay. Anti-C. baileyi IgG antibody levels remained high (1:106.67 to 1:512.00) for at least 4 months with 330 days of a detectable period. Ten days after the negative conversion, each chicken was re-challenged with 1 x 10(7) oocysts of the same species. Subsequent infection in 340-day-old individuals caused sudden elevated IgG antibody levels and the titer peaked on day 28 postchallenge inoculation (PCI), at 1:1.024 with a 65 days of detection period. Chickens in primary infection showed oocyst shedding profiles, but did not exhibit any oocyst shedding before or after experimental reinfection.     

The survival of inoperable breast cancer treated with radiotherapy and radiotherapy followed by mastectomy

A 6 1/2-day prepatent period and a patent period of at least 22 days followed single oocyst infection of a young coccidia-free hare with Eimeria robertsoni. Size of oocysts increased significantly during patency and was negatively correlated with oocyst output. Oryctolagus cuniculus remained negative after inoculation per os of E. robertsoni of snowshoe hare origin.     

Effect of aging on survival and pathogenicity of Eimeria acervulina and Eimeria acervulina and Eimeria tenella

After 6 to 8 months of storage, cultures of sporulated Eimeria acervulina and E. tenella oocysts ahd a marked drop in the number of sporocysts that survived grinding, sporozoites that survived after excystation, and sporozoites that penetrated chick kidney cells in vitro. The rate of excystation was unaffected by storage of up to one year. In vivo pathogenicity, based on weight gain, lesion score, and plasma pigment, declined after 5 months of oocyst storage. The reduction in pathogenicity in vivo could be compensated for by adjusting the inoculation dosage to reflect the loss of infectivity seen in the in vitro test.     

Effect of tuberculosis on milk production in dairy cows

A survey of Isospora suis performed in 177 faecal samples from 30 swine farms detected thin wall type I. suis oocysts in seven samples. This type of oocyst measuring 23.9 by 20.7 microns had a retracted thin wall similar to that of the genus Sarcocystis. This type of oocysts, isolated from four different faecal samples, was inoculated in four-five-days-old piglets free of contamination in order to verify the life cycle and pathogenicity of the species. The pigs were kept in individual metal cages and fed with cow milk. Daily faecal collections and examinations were performed until the 21st day after infection. MacMaster and Sheather's methods were used for oocyst counting and identification. Infected piglets produced yellowish-pasty diarrhoea with slight dehydration. The prepatent and patent periods were respectively from 6 to 9 and 3 to 10 days after infection. Oocyst elimination was interrupted on the 10th and 11th days after infection with biphasic cycles. Thin and thick wall oocysts were detected in the same faecal samples. Thin walls were not observed in unsporulated oocytes. The observations suggest that this type of oocysts could appear in specific strains which occur in the later stages of their development. These oocysts seem to be responsible for clinical and pathogenic signs of neonatal isosporosis in pigs.     

Multiple pathways lead to activation of the survival mechanism in quiescent BALB/c-3T3 cells

We investigated the effects of human anti-sporozoite antibodies on the sporogonic development of Plasmodium falciparum in Anopheles stephensi. Equal volumes of washed human erythrocytes and human sera from 1) volunteers with protective immunity induced by immunization with irradiated P. falciparum sporozoites, 2) the same volunteers before immunization, or 3) Kenyans exposed to natural sporozoite transmission, were fed to cohorts of P. falciparum-infected A. stephensi on either day 5, 8, or 11 after infection. A fourth group of infected mosquitoes from the same cohort were not refed. In two experiments, the effects of anti-sporozoite antibodies were evaluated by determining the infection rates and parasite densities for oocysts and salivary gland sporozoites. There was no evidence that anti-sporozoite antibodies had any effect on the development or intensity of P. falciparum infection in A. stephensi. However, accelerated oocyst maturation was associated with mosquitoes taking a second blood meal, independent of serum source. Salivary gland sporozoites from mosquitoes that fed on immune human sera contained bound human IgG, which was detectable by indirect immunofluorescence assay. The infectivity and transmission potential of human IgG-coated sporozoites is unknown.     

IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in gut-associated lymphoid tissue during resolution of infection

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.     

Mapping for B-cell epitopes in the GX3262 antigenic sequence derived from Eimeria tenella sporulated oocysts

Overlapping heptapeptides derived from the MA16 Eimeria acervulina antigenic sequence (Castle et al., 1991) were synthesised on polypropylene pins ('pepskan' technique, Cambridge Research Biochemicals, UK). Binding of antibodies from chickens and rabbits infected and immunised respectively with various species of Eimeria oocysts (E. acervulina, E. tenella, E praecox, E. necatrix and E. maxima), was examined using the coated pins as the solid phase of an enzyme immunoassay (EIA). Antigenicity of the overlapping synthetic heptapeptides was then analysed using a number of algorithms based on the amino acid sequence to predict secondary protein structure, hydrophilicity, acrophilicity and chain flexibility profiles. The antigenicity of this sequence appears to be quite different from that found for the E. tenella GX3264 antigenic sequence (Bhogal et al., 1992) whose profile was similarly examined (Talebi and Mulcahy, 1994) using the same rabbit and chicken anti-Eimeria oocyst sera.     

Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.     

Transformation of bisphosphonates into insoluble material in human blood in vitro

The localization and duration of developmental stages of Eimeria colchici and Eimeria duodenalis were studied histologically. The prepatent period of the most pathogenic species from the caeca of pheasants--Eimeria colchici--was 6 days. The patent period began on the 7th day and finished on the 11th day post-infection with the maximum production of oocysts on days 8-9. In the case of Eimeria duodenalis the prepatent period was shorter--4 days, and the duration of the patent period was 3-4 days without a significant increase in oocyst production.     

Simulating the Effect of Liquid CO2 on Cryptosporidium parvum Oocysts in Aquifer Material

The effects of liquid CO2 injection on the viability of Cryptosporidium parvum oocysts were evaluated. A laboratory study was designed to test the effects of saturated CO2, freeze–thaw cycles and different freezing protocols on C. parvum oocysts in aquifer material. Oocysts were exposed to a saturated solution of CO2 at room temperature for 1-, 4-, 8-, and 12-h intervals and their viability was compared with controls. One- and three-cycle freeze–thaw experiments on oocyst survival were conducted. Inactivation of oocysts was assessed for: (1) rapid freezing and rapid thawing and (2) gradual freezing and rapid thawing. Exposure to 1 atm of CO2 in water at room temperature had a negligible effect on oocyst viability. Average oocyst viability after the one- and three-cycle freeze–thaw experiments was 24.7 and 2.7%, repsectively. The average oocyst viability associated with the rapid freeze–thaw and gradual freeze–thaw experiments was 11.3 and 26.2%, respectively. Freezing associated with injection of liquid CO2 into aquifers would be the factor inactivating oocysts; to cause a 3-log decrease in oocyst viability multiple injections may be required.     

Effects of combined treatment with recombinant bovine somatotropin and immunization with live oocysts on performance of broiler chicks raised in coccidia-seeded floor pens

In our laboratory, preliminary studies have indicated that recombinant bovine somatotropin (rbST) can stimulate protective immunity against coccidia infection. A floor pen trial on coccidia-seeded litter was run to further test its activity as an adjuvant during immunization of chicks with a live oocyst vaccine. Five hundred day-old male broiler strain chicks were randomly assigned to five experimental Treatments: 1, medicated controls; 2, unimmunized, not treated with rbST; 3, unimmunized, rbST-treated; 4, immunized, not treated with rbST; 5, immunized, rbST-treated. Each treatment consisted of four pens of 25 chicks each. At the end of the growout period (7 wk), the chicks in Treatment 1 (medicated controls) had the highest mean BW, but mean BW of chickens in Treatment 3 (rbST treatment only) were not significantly less. On the other hand, the mean weights of chicks in Treatments 4 (immunized only) and 5 (immunized plus rbST) were significantly reduced, and not different from those of the untreated chickens (Treatment 2). However, when challenged at 3 wk, the chicks in Treatment 5 had a mean combined total lesion score that was significantly lower than that from Treatment 3, indicating that they had developed a higher degree of specific immunity, but of the expense of weight gain. The results suggest that rbST has a potential for use as an adjuvant with live oocyst vaccination, but that the ratio between rbST dose and numbers of oocysts in the live vaccine needs to be carefully controlled.     

Kinetics of specific immunoglobulin A, M and G production in the duodenal and caecal mucosa of chickens infected with Eimeria acervulina or Eimeria tenella

The development and appearance of antibody was studied in the intestine and serum from histocompatible GB1 chickens orally infected with oocysts of Eimeria acervulina (restricted to the duodenum) or Eimeria tenella (restricted to the caeca). The local immune response was measured as the specific antibody levels in the supernatants of intestinal fragments (duodenum and caecum) maintained in culture for 16 h at 41 degrees C, 5% CO2, 95% air. Specific IgM was detected 1 week after E. acervulina infection, and the specific IgA and IgG contents of the duodenum and caecum were significantly elevated (P < 0.001) after 2 weeks. The intestinal specific IgG content was raised. E. tenella infection resulted in specific IgA only in the parasitized area during the second week post-infection (P < 0.05). Specific IgM and IgG were both detected in the duodenum and caecum, respectively, 1 and 2 weeks p.i. Production of parasite-specific immunoglobulins was always significantly higher in the parasitized than in the unparasitized areas (caeca for E. acervulina, duodenum for E. tenella). This ex vivo culture assay of intestinal fragments used to measure the mucosal immune response of intestinal areas showed a significant production of specific IgA and IgM. In addition, high levels of IgG were also measured. The role of this specific IgG in Eimeria infection remains to be determined.     

Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum

Bovine hyperimmune anti-Cryptosporidium colostrum immunoglobulin (BACI) decreases the intensity of Cryptosporidium parvum infection in vitro. We investigated the prophylactic effect of BACI in healthy adults challenged with C. parvum. After we established an oocyst dose that resulted in 100% infection in four volunteers (baseline group), 16 volunteers were randomized to receive (1) BACI prior to C. parvum challenge (BACI group) and a nonfat milk placebo 30 minutes later, (2) BACI prior to and 30 minutes after challenge (reinforced BACI group), or (3) nonfat milk placebo prior to and 30 minutes after challenge. Subjects received BACI (10 g) or nonfat milk placebo three times a day for a total of 5 days and were followed for clinical symptoms and oocyst excretion for 30 days. A trend toward less diarrhea (P = .08) was observed for subjects receiving BACI in comparison with occurrences in placebo recipients. Subjects receiving BACI or nonfat milk placebo had a 100-fold reduction in oocyst excretion as compared with excretion in the baseline group.     

Child psychiatry. The family: a valuable partner]

Plasma chemistry and haematological studies were conducted on chickens with coccidiosis. Male White Leghorn chickens, of two weeks old, were inoculated with 5 x 10(4) Eimeria tenella sporulated oocysts or with 1 x 10(6) E acervulina sporulated oocysts. Blood samples were taken four, seven and 11 days after inoculation. A wet chemistry system was applied to measure the plasma activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, amylase and lactate dehydrogenase and the concentrations of creatine, total bilirubin, urate, total cholesterol, total protein, albumin, glucose and triglycerides. A dry chemistry system was applied to measure sodium, potassium, chloride and calcium. The number of red blood cells and packed cell volume were determined by a micro cell counter and blood pH was measured with a blood gas analyser. The erythrocyte count, packed cell volume, sodium and chloride levels in the chickens infected with E tenella were significantly (P < 0.05) lower than those of the uninfected controls. The significant decrease in blood pH of the chickens infected with E acervulina suggests malabsorption associated with duodenal lesions induced by the infection.     

A preliminary study of the nature of infection and immunity in chickens given an attenuated line of Eimeria acervulina

The Houghton (H) strain of Eimeria acervulina was attenuated by serial passage through chickens of the first oocysts produced during infection. This selection pressure resulted in a reduction in the pre-patent period of the parasite, shown to be due to the selection of a line predominantly with only 3 instead of 4 generations of schizonts. The precocious line had a reproductive potential much lower than that of the parent strain and it was significantly less pathogenic. Chickens given oocysts of the precocious line were almost completely immune to challenge with the Houghton strain.