Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens

Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5–10 μg antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.     

Recombinant bovine interleukin-12 stimulates a gut immune response but does not provide resistance to Cryptosporidium parvum infection in neonatal calves

This study was undertaken to determine if administration of recombinant bovine interleukin-12 (rBoIL-12) could stimulate a cellular immune response that protected calves from an oral challenge inoculation with Cryptosporidium parvum oocysts. In a first experiment, rBoIL-12 intraperitoneally administered as a single dose 1 day before challenge inoculation, did not alter the course of infection. The percentage of immune competent cells and levels of cytokine gene expression in the ileo-cecal mucosa and in the draining lymph nodes of treated calves were similar to those of untreated control calves. However, when rBoIL-12 was subcutaneously administered daily from 2 days before infection to 2 days after infection, a consistent increase of T lymphocytes and an higher expression of interferon-gamma (IFN-gamma) was detected. Again, treatment did not alter the course of infection. Similar results were obtained when rBoIL-12 was administered daily for 4 days beginning 2 days after oral inoculation. These data indicate that although rBoIL-12 stimulated a strong immune response in the gut of neonatal calves, the response was not able to provide protection from challenge inoculation with C. parvum oocysts.     

Eimeria tenella in chickens: Studies on resistance to the anticoccidial drugs monensin and lasalocid

The response of the Houghton strain of E. tenella to monensin and lasalocid is described. Neither drug was able to completely control this strain at 125 ppm. At this concentration oocyst production was not completely suppressed by monensin in birds given ten oocysts. At higher concentrations both drugs were able to control E. tenella (H) in birds given 1 × 10⁵ oocysts but monensin was toxic. 375 ppm monensin did not prevent oocyst production in birds given 4 × 10⁶ oocysts. Resistance could not be developd to either of these compounds after 12 serial passages in the presence of drug. Serial passage of E. tenella (H) in the presence of monensin however resulted in an increase in pathogenecity. This greater pathogenecity was not due to a better ability to multiply in the host.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants.

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

The Effect of Dietary Crude Protein Level on Intestinal and Cecal Coccidiosis in Chicken

The effect of interaction of crude protein level in the diet and coccidiosis of the cecum and small intestine of chicks was investigated. A total of 390 day-old chicks were divided in 36 groups of ten and six groups of five chicks each. Twelve groups of ten and two groups of six chicks each were fed one of the three diets based on dietary crude protein level (16%, 20% and 24%). All diets contained an equal energy concentration. The chicks were on the appropriate diet for 15 days prior to infection. Each group was then subjected to one of the three treatments (a) control, (b) a single dose infection with 100,000 oocysts of Eimeria acervulina and (c) a single dose infection with 10,000 oocysts of Eimeria tenella. On the eighth day post infection all surviving E. tenella infected chicks and two replicates per dietary treatment of control and E. acervulina infected chicks were killed. An increase in dietary crude protein led to a linear (P<0.01) increase in daily gains and feed efficiency but did not affect feed consumption of chicks during one to 15 days pre-infection. Coccidiosis caused a reduction in daily gain, feed consumption and efficiency of feed utilization, the effect being more severe in E. tenella infection. The effect of dietary crude protein was protective against weight reduction. Chicks infected with E. tenella fed 24% crude protein had a higher (P<0.01) mortality rate than those fed on 16% or 20% crude protein level. The oocyst production by E. acervulina infected chicks was also higher (P<0.01) at the 24% crude protein level. The E. acervulina infected chicks exhibited compensatory growth during the eight to 14 days post infection. The compensatory growth was superior at the higher crude protein levels. The mechanism of compensatory growth is discussed.     

Effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice

OBJECTIVE: To evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus. ANIMALS: 2,213 mice. PROCEDURE: Inactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus. RESULTS: Vaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.     

Local immunity in the respiratory tract of the chicken. II. The secretory immune response to newcastle disease virus and the role of IgA

The nature, specificity and characteristics of the secretory immune response in the respiratory tract of the chicken were investigated in young specific-pathogen-free chickens after vaccination with live lentogenic and inactivated Newcastle disease virus (NDV). Virus-neutralizing (VN) activity considerably exceeding transudation levels from serum were detected in lachrymal fluid, saliva and tracheal washes following infection by both ocular and oral routes. Heat inactivated virus inoculated into the trachea evoked neither serum nor secretory VN activity, whereas a commercial inactivated virus vaccine in mineral oil adjuvant stimulated high titres of serum antibody and some VN activity in tracheal fluids.Antibody in secretions limited, but did not prevent, reinfection of the trachea when birds were challenged 2 weeks later. In contrast to an elevation of circulating antibody titre, challenge induced only a repeated primary response of secreted antibody.All secretions contained IgA which, at least in saliva, accounted for 85% of its activity, the remainder being due to IgG. Fluorescent localization of immunoglobulin producing cells (IPC) demonstrated large numbers containing IgA in association with the upper respiratory tract, particularly in the Harderian gland which contained dense aggregations of plasma cells, many of which were producing IgA.It is concluded that the respiratory tract of the chicken possesses an antibody mediated secretory immune system analogous to that of mammalian species.     

Immune responses of sheep to louping-ill virus vaccine

The immune responses of sheep to single and double doses of commercially available louping-ill virus vaccine were examined. The susceptibility to challenge of sheep which had been vaccinated but showed a poor response was also investigated. Two injections of vaccine were required to provoke an adequate antibody response and maximum titres were obtained when there was an interval of two to eight weeks between injections. After challenge, viraemia could not be detected in animals with an antibody titre of 20 although increase in the concentration of humoral antibodies indicated that infection had occurred. Vaccinated but seronegative sheep and vaccinated animals with an antibody titre of 10 were also clinically resistant to the challenge, although circulation of virus was demonstrated. That vaccination had sensitised those animals to viral antigen was evident from the reduced viraemias, the early rise in humoral antibody titres and subsequent protection afforded compared to unvaccinated control animals. Thus, animals with minimal antibody titres after vaccination are protected, but it is recommended that vaccines eliciting the highest possible antibody responses will be the most useful under field conditions.     

Immunizing potential of various stages of Taenia ovis eggs injected subcutaneously into neonatal or sixteen-week-old lambs

When viable eggs of Taenia ovis were given orally to 1-week-old lambs, infection occurred only in those lambs that had been deprived of colostrum. When viable eggs were injected subcutaneously into 1-week-old lambs, no larvae developed at the injection site, and no resistance was stimulated against an oral challenge of T. ovis eggs given 11 weeks later. However, when eggs, oncospheres or developing cysticerci were subcutaneously injected into 16-week-old lambs, all grew at the injection site and stimulated a high degree of immunity to oral infection. Colostrum-derived antibodies against T. ovis apparently suppressed the immunizing potential of T. ovis eggs injected subcutaneously into neonatal lambs.     

Effect of heat killed Mycobacterium phlei on body weight gain and management of caecal coccidiosis in broiler chickens

The objective of this study was to evaluate the immunotherapeutic potential of heat killed Mycobacterium phlei in broiler chicken against experimentally produced Eimeria tenella infection. The selected dose of E. tenella oocyst (5 × 10³ sporulated oocysts per bird) was capable of producing a mild form of caecal coccidiosis as observed by significant difference in body weight gain, clinical findings and caecal lesion score. Heat killed M. phlei was fed orally at 10 mg per bird with sterile PBS vehicle at alternate day for four doses. Our study reveals that per day body weight gain was significantly (p < 0.01) higher for healthy control compared to coccidia infected group. The group fed M. phlei along with coccidial challenge showed significantly (p < 0.05) higher body weight gain than infected control group. Heat killed M. phlei feeding also found effective to reduce the caecal lesion score significantly (p < 0.05) in comparison to E. tenella infected untreated group. IgA concentrations in serum and bile at 7-day post challenge of coccidial oocyst was also significantly (p < 0.01) higher in M. phlei fed group when compared to coccidia infected and healthy control group. We concluded that use of heat killed M. phlei has a beneficial role as an immunostimulant against caecal coccidiosis in broiler chicken.     

Influence of low levels of dietary aflatoxins on Eimeria tenella infections in broilers

The present study was conducted to evaluate the adverse effects of an interaction between low levels of dietary aflatoxins (AF) and Eimeria tenella infection on broiler chicks. A set of 1-day-old chicks were raised for 35 days in the following groups: a control group, a group fed AF, a group fed AF and inoculated with E. tenella (AF + E.ten), and a group inoculated with E. tenella alone. AF in the contaminated diet were given at 200 ppb starting from the seventh day after hatching while E. tenella was inoculated at a dose of 5 × 10⁴ sporulated oocysts per chick at the 14th day after hatching. Worsened performance traits and high mortality were all observed in the treated birds, particularly the AF + E.ten group. Lesion scores and oocyst outputs were not different within groups. Chickens fed with AF had significantly increased serum ALT and ALP activities as well as decreased albumin content. They also showed hepatomegaly, hepatocytic vacuolation and necrosis, an atrophied bursa of Fabricius, and a thymus with tissue depletion. E. tenella-infected broilers displayed a significant reduction in packed cell volume, hemoglobin content and lymphocyte percentage, and showed hemorrhagic typhlitis. The deficits in hepatic function and hematologic parameters as well as the gross pathological, and histopathological changes, were more common and more severe in the group that was exposed to both aflatoxicosis and coccidiosis than in the groups exposed to either treatment alone. Thus, the combination of aflatoxicosis and E. tenella infection may influence the course of coccidial infection due to additive effects.     

A comparison of the enzyme linked immunosorbent assay and the indirect haemagglutination technique applied to sera from cattle experimentally infected with Taenia saginata (Goeze, 1782)

The serological response of 6 calves to experimental oral infection with between 60,000 and 100,000 Taenia saginata eggs at 3–12 months of age was monitored by the enzyme-linked immunosorbent assay (ELISA) and the tanned cell indirect haemagglutination technique (IDH). A serum antibody response was detected by both techniques by 2–3 weeks post infection, rising to a plateau about 4–6 weeks post infection. The serum antibody levels began to decline by about 30 weeks post infection. Two uninfected control cattle gave negative reactions.In addition, the serological response of 5 calves which had received a dose of 10,000 T. saginata eggs at 2–3 days of age and then weekly serial doses of 500 eggs for 12 months thereafter, was compared with a similar group of 5 calves, which had received the single infection of 10,000 eggs at 2–3 days of age only. Calves in both groups developed an antibody response detectable by the ELISA technique whereas those in a group of 5 control calves did not show such a response. When studied individually however there was marked variation in the serum antibody levels of these young cattle, as although some calves gave a relatively strong serological response, others hardly varied from the controls.     

THE INDIRECT HAEMOLYSIS TEST (IHLT) FOR BOVINE BRUCELLOSIS—COMPARISONS WITH THE COMPLEMENT FIXATION TEST (CFT) IN VACCINATED AND EXPERIMENTALLY INFECTED CATTLE

SUMMARY Cattle were vaccinated with Brucella abortus strain 19 subcutaneously in doses ranging from the normal (2.0 ml) dose of standard vaccine down to 1/400 of the normal dose, and via the conjunctival sac with 1/2 or 1/20 of the normal dose. Under all vaccination regimes serum antibody titres in the complement fixation test (CFT) rose more rapidly, reached higher levels, declined more slowly and involved a greater proportion of animals, than titres in the indirect haemolysis test (IHLT). The interval between the first positive serological test and parturition was determined for 54 pregnant cows infected with a virulent field strain (VRI 3) of B. abortus. On average the CFT titre rose to 1/4 43 days, and 1/8 33 days, before parturition, while the IHLT reached a 2/8 reaction 31 days before parturition.     

The pathology and pathogenesis of Salmonella stanley infection in experimental chicks

Experimental infection with Salmonella stanley was produced by oral, intravenous and intramuscular routes in day-old chicks. The earliest evidence of the presence of the organisms was in duodenal mucosa six hours after oral infection. Following oral infection the organisms were detected in the duodenum from six hours to five days, in the caecum from 12 hours to nine days, liver, spleen and blood from 24 hours to seven days. The resistance to infection was found to be significant after 10 days old, but not up to six days old. The work confirmed that the survival time of birds given S stanley by the intravenous or intramuscular routes was inversely proportional to the dose up to a maximum beyond which the survival time was not further decreased by dose increase. The presence of S stanley in tissues and blood was detected by isolation and by the fluorescent antibody technique.     

The passive protection of lambs against Clostridium perfringens type D with semi-purified hyperimmune serum

Weaned lambs, having a detectable level of maternal antibodies (1-2 units/ml) against C. perfringens type D, showed protective antitoxin levels lasting for 29 days after receiving a single parenteral dose of 200 units/kg hyperimmune serum. Lambs, having no maternal antibodies (less than 0,07 units/ml) to C. perfringens type D but receiving the same dose of hyperimmune serum, maintained protective antibody levels for only 21 days. Three weeks after the titres fell below the minimum protective level of 0,15 units/ml, both these groups were treated again in the same manner. The passive immunity conferred in both groups now lasted for 42 days. When the hyperimmune serum was administered to lambs already immunized by vaccination, a slight increase was noted in the antibody titre.     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Adjuvanted Outer Membrane Protein Vaccine Protects Poultry against Infection with Salmonella enteritidis

The immunogenicity of a sonicated extract (SE) and of outer membrane proteins (OMP) of Salmonella enteritidis was tested in birds of about 8 weeks of age. The dose, route of vaccination and the adjuvant used varied in different groups of birds. Two vaccine doses with or without adjuvant were given parenterally or orally 3 weeks apart. OMP vaccines gave significantly higher antibody titres than SE vaccines, as indicated by ELISA. The vaccines adjuvanted with oil produced higher antibody titres than those without any adjuvant. A dose of 1 mg of vaccine produced higher antibody titres than 0.5 mg of vaccine. Adjuvanted vaccine given subcutaneously elicited higher antibody responses than oral vaccines given without adjuvant. The birds were challenged with virulent S. enteritidis organisms at the end of the second week after a booster dose. None of the birds given 1 mg of OMP vaccine subcutaneously shed the organisms when tested by culturing cloacal swabs, although a few birds vaccinated with 0.5 mg of OMP vaccine did so. In general, adjuvanted OMP vaccines gave better protection than SE vaccines.     

Demonstration of antibodies to Eimeria species in lambs by an enzyme-linked immunosorbent assay

Soluble extracts were prepared from sporulated oocysts, unsporulated oocysts and merozoites of Eimeria crandallis, E faurei and E ovinoidalis. They were assessed for antigenicity and specificity by ELISA using rabbit antisera to sporulated oocysts or merozoites. Antibody levels were examined in sera from colostrum-deprived coccidia-free lambs, conventionally reared lambs and lambs which had received experimental infections. Maternal antibody was demonstrated in colostrum and in serum taken at 24 hours from all conventionally reared animals but not colostrum-deprived animals. Antibody levels in conventional animals dropped over the first five weeks of life and rose again during the next five weeks. Antibody was not detected in coccidia-free animals. Monospecific infections of E faurei or E ovinoidalis demonstrated antibody responses to primary and secondary infections. Some specificity of response was suggested with E faurei infection. The antigen preparations showed considerable cross-reactions between species. These serum antibody responses, although appearing too late for individual diagnosis, may assist diagnosis on a flock basis.     

Seroprevalences and lack of abortions after vaccination of Churra sheep with reduced doses of Rev. 1 Brucella melitensis vaccine by subcutaneous or conjunctival routes

A field study to evaluate the serological response and the safety of different doses and administration routes of the Rev. 1 vaccine was carried out on two Churra breed flocks. Reduced doses of 2.3 × 10⁶ and 3 × 10⁷ live organisms were administered by the subcutaneous or the conjunctival route, respectively. In those animals which were seropositive before vaccination, the percentage of positive sera declined progressively in a similar way in all groups over the 36 weeks that the study lasted; the antibody titers also dropped continuously in the group vaccinated by the conjunctival route with the lower dose, while in the remaining three groups there was a transitory increase in the 4th week after vaccination. In those animals which were serologically negative prior to vaccination, the percentage of positive sera and the antibody titers generally reached their peak in the 4th week after vaccination, followed by a progressive decline in succeeding weeks. Similarly, titers were higher in animals vaccinated subcutaneously than in those vaccinated by the conjunctival route. The differences between the frequencies of positive sera and the levels of antibodies were important when routes were compared. Animals receiving a dose of 2.3 × 10⁶ CFU subcutaneously had a satisfactory serological response, with a more rapid decline in their level of antibodies than in the animals which were vaccinated with 3 × 10⁷ CFU by the same route. No cases of abortion were reported in the 461 vaccinated ewes.