Zinc bacitracin enhances colonization by the intestinal spirochaete Brachyspira pilosicoli in experimentally infected layer hens

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 birds were sham-inoculated with sterile broth. All chickens received a commercial layer diet, but 10 infected birds had 50 parts/10 6 zinc bacitracin (ZnB) incorporated in their food. Birds were kept for 7 weeks, and faecal moisture, egg numbers, egg weights and body weights were recorded weekly. B. pilosicoli was isolated from the faeces of only three of the 30 inoculated birds receiving the diet without ZnB, whereas seven of the 10 inoculated birds receiving ZnB in their diet were colonized. This difference in colonization rate was highly significant ( P = < 0.001). Dietary ZnB at 50 parts/10 6 therefore predisposed to colonization by B. pilosicoli. Despite colonization, no significant production differences were found between the birds in the three groups.     

Influence of in-feed zinc bacitracin and tiamulin treatment on experimental avian intestinal spirochaetosis caused by Brachyspira intermedia

Thirty individually caged layer hens were inoculated with Brachyspira intermedia , and 20 control birds remained unchallenged. Birds received a diet containing 100 parts/10 6 zinc bacitracin (ZnB), and were monitored for 10 weeks. B. intermedia was recovered sporadically from five of the inoculated birds, and there were no significant effects on body weight, faecal water or egg production. ZnB was presumed to be indirectly inhibiting spirochaete growth, and when removed from the diet, 18 of the 30 inoculated birds rapidly became culture positive. After 4 weeks, 10 of the 30 infected birds were treated with tiamulin at 25 mg/kg for 5 days, and 10 were returned to the diet containing ZnB. Birds receiving tiamulin became spirochaete negative and maintained their egg production, but re-infection occurred. The other 20 infected birds had a significant drop in egg production, but those receiving ZnB showed a reduced colonization by B. intermedia after 3 weeks.     

Evaluation of tiamulin and lincomycin for the treatment of broiler breeders experimentally infected with the intestinal spirochaete Brachyspira pilosicoli

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 30 individually caged 22-week-old Cobb 500 broiler breeder hens. Another 10 birds were sham-inoculated with sterile broth. All birds failed to become colonized. At 29 weeks of age, all birds were transferred to a diet containing 50 parts/10 6 zinc bacitracin (ZnB) and were re-challenged with the same B. pilosicoli strain at 32 weeks of age, weekly for 5 weeks. The majority of the inoculated birds then became colonized, confirming previous findings that ZnB can increase susceptibility to colonization with B. pilosicoli. The control group remained uninfected. Infected groups tended to have an increased faecal water content and faecal staining of eggshells. Ten birds were then treated by crop tube with 25 mg/kg body weight tiamulin for 5 days, and 10 birds with 20 mg/kg body weight lincomycin for 5 days. Both treatments removed the infection, while untreated birds remained infected. The results support previous observations that ZnB at 50 parts/10 6 in the diet increases the susceptibility of birds to B. pilosicoli infection, and demonstrated the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.     

Prevalence,disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.     

Cloning and DNA sequence analysis of an immunogenic glucose-galactose MglB lipoprotein homologue from Brachyspira pilosicoli, the agent of colonic spirochetosis

Colonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirochete Brachyspira (formerly Serpulina) pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of human B. pilosicoli strain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the cloned B. pilosicoli gene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putative mglB gene was a 3'-truncated open reading frame with 73.8% similarity and 66.3% identity to mglA of Escherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present in B. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetes Serpulina intermedia, Brachyspira innocens, Brachyspira murdochii, and the avian Brachyspira alvinipulli, but not in porcine Brachyspira hyodysenteriae, human Brachyspira aalborgi, and porcine Treponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [(3)H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis of B. pilosicoli in vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis.     

Differentiation of porcine Brachyspira species by a novel nox PCR-based restriction fragment length polymorphism analysis

A novel PCR-based restriction fragment length polymorphism analysis of the Brachyspira nox gene was developed. The restriction patterns for Brachyspira hyodysenteriae, B. pilosicoli, B. intermedia, B. murdochii, and B. innocens were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust.     

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens.

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n = 25) and Brachyspira pilosicoli (n = 17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC > 4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC > 32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.     

Treatment of a field case of avian intestinal spirochaetosis caused by Brachyspira pilosicoli with tiamulin.

There has been much confusion over the significance of spirochaetes found in the caeca of laying hens and the impact they may have on egg production. In recent years, the situation has been made clearer and the presence of such species as Brachyspira pilosicoli have been shown to cause a mild, chronic disease in both layers and breeders and to reduce egg production by reportedly 5%. In the United Kingdom, a multi-age caged laying site with three separate flocks of approximately 12 000 birds each was chronically infected with B. pilosicoli but displayed few clinical signs except for a noticeable reduction in egg production and an increased mortality. The flocks were treated for 3 days in the drinking water with tiamulin at 12.5 mg/kg bodyweight, and a steady improvement in performance was recorded. The production results were compared with a flock that had been untreated with tiamulin previously, as a control, and one that had been treated at 25 and 45 weeks of age. A 9.8% improvement in egg production/hen housed up to 72 weeks of age and 9.7% in total egg weight was recorded, as well as an 8.6% reduction in actual hen mortality, in the tiamulin-treated flock in comparison with the untreated control. After taking into account the difference in breeds used, there was only a 6% reduction in egg production but an 8.84% increase in mortality in the untreated flock compared with the individual breed's standard production data. The cost of the disease was estimated at 14 million pound in the United Kingdom, based on a national laying flock of 30 million or 1.5% of production. Faecal examination for potentially pathogenic spirochaetes should be part of the differential diagnosis of under-performing laying flocks.     

Intestinal spirochaetes (Brachyspira spp.) colonizing flocks of layer and breeder chickens in Malaysia

Avian intestinal spirochaetosis causes problems including delayed onset of lay and wet litter in adult chickens, and results from colonization of the caecae/rectum with pathogenic intestinal spirochaetes (genus Brachyspira). Because avian intestinal spirochaetosis has not previously been studied in South East Asia, this investigation was undertaken in Malaysia. Faecal samples were collected from 25 farms and a questionnaire was administered. Brachyspira species were detected by polymerase chain reaction in 198 of 500 (39%) faecal samples from 20 (80%) farms, including 16 (94%) layer and four (50%) breeder farms. Pathogenic Brachyspira pilosicoli was identified in five (29%) layer and two (25%) breeder farms whilst pathogenic Brachyspira intermedia was detected in nine (53%) layer and one (12.5%) of the breeder farms. Twelve (80%) layer farms had egg production problems and 11 (92%) were positive for Brachyspira: three (25%) for B. pilosicoli and six (50%) for B. intermedia. Of three breeder farms with egg production problems, one was colonized with B. pilosicoli. Three of ten layer farms with wet litter were positive for B. pilosicoli and six for B. intermedia. Of four breeder farms with wet litter, one was colonized with B. pilosicoli and one with B. intermedia. No significant associations were found between colonization and reduced egg production or wet litter, perhaps because so many flocks were colonized. A significant association (P = 0.041) occurred between a high prevalence of colonization and faecal staining of eggs. There were significant positive associations between open-sided housing (P = 0.006), and flocks aged >40 weeks (P < 0.001) and colonization by pathogenic species.     

Vaccination of chickens with the 34 kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100?μg (B) or 1?mg (C) protein emulsified with Freund’s incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1?mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P?=?0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections.

RESEARCH HIGHLIGHTS

• Laying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72.

• Immunized birds had a highly significant reduction in the duration of colonization.

• Fewer immunized than control birds had abnormal caecal contents after infection.

• Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

     

Dual infection with Mycoplasma synoviae and a tenosynovitis-inducing reovirus in chickens

One group of embryonated chicken eggs was inoculated with sterile myco-plasma broth and another with a broth culture of Mycoplasma synoviae. Each group was subdivided into two and the chicks were hatched in four isolation pens. At 1 day of age one uninfected and one M. synoviae infected group were inoculated with a reovirus known to cause tenosynovitis. Birds were closely observed, and some were killed for sampling as chicks (1-5 weeks) and the remainder as adults (25-30 weeks). Clinical signs related to both organisms were seen in the dual-infected chicks and were more pronounced than in either of the corresponding single infections. There were significant weight differences between the infected and control chicks, except in single M. synoviae infection, and the mean weight of the birds with mixed infection was less than those with either single infection. Gross and histological lesions were observed in the hocks and livers of chicks in each infected group although with a higher incidence and severity in the dual-infection. Microscopic lesions were seen in the hearts of some birds in all three groups. Leg lesions associated with the reovirus infection persisted to maturity and another observation in mature, virus-infected birds was impaired egg production and fertility. M. synoviae was reisolated consistently from tracheas of single and dual-infected birds of all ages and reovirus from the tendons of inoculated birds for 4 weeks. Antibodies to both agents persisted throughout the period of the experiment.     

Pathogenicity of latent infectious laryngotracheitis virus in chickens

Groups of specific pathogen-free chickens were inoculated with the same dose of a field strain of infectious laryngotracheitis virus that had either been isolated from tracheal swabs taken from infected birds during acute phase shedding, or that had been isolated during an episode of virus shedding after a latent period of 12 to 17 weeks. Birds in both groups developed characteristic clinical signs of ILT including difficulty in breathing, rales and sneezing. Thus, ILT virus shed after a latent period is capable of causing disease in susceptible birds similar to that seen in the field.     

Comparative prevalences of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli as etiologic agents of histologically identified intestinal spirochetosis in Australia

DNA from gastrointestinal biopsy specimens from 28 Australian patients with histologic evidence of intestinal spirochetosis (IS) was subjected to PCRs to amplify segments of the 16S rRNA and NADH oxidase genes of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli. B. aalborgi was identified in specimens from 24 (85.7%) patients and B. pilosicoli in those from 4 (14.3%) patients (2 of whom were also positive for B. aalborgi). For two patients, no product was amplified. This study demonstrates that B. aalborgi is much more commonly involved in histologically identified IS in Australian patients than is B. pilosicoli. This is the first report of amplification of B. pilosicoli DNA from humans with IS.     

Vaccination against histomonosis prevents a drop in egg production in layers following challenge

The effect of attenuated Histomonas meleagridis on pullets was investigated and the protection of vaccinated adult laying hens against a severe challenge was studied in the same experimental setting. Four groups of 25 pullets were set up at 18 weeks of life and birds in two groups were vaccinated with in vitro-attenuated H. meleagridis. Chickens in two groups (vaccinated and non-vaccinated) were challenged 5 weeks later with virulent histomonads, while the remaining groups were retained until termination of the study 11 weeks post vaccination. Vaccination of pullets did not have any impact on their subsequent performance. Egg production of non-vaccinated but challenged birds dropped significantly (P≤0.05) between 2 and 4 weeks post challenge (p.c.) to 58.7%, compared with 90% in control chickens. At 4 weeks p.c., the drop in egg production in vaccinated and challenged birds was significantly lower (P=0.02) than in non-protected layers. Pathological changes were found only in challenged birds 2 and 6 weeks p.c. Several non-vaccinated birds showed severe lesions in the caeca with sporadic involvement of the liver and atrophy of the reproductive tract. Vaccination prior to challenge reduced the incidence of pathological findings. For the first time, vaccination of pullets with in vitro-attenuated histomonads could be shown to be an effective and safe prophylactic tool to prevent a severe drop in egg production of commercial layers following experimental infection.     

Age related resistance to avian leukosis virus. II. Influence of age at inoculation on mortality and congenital transmission

Six age groups of specified pathogen free White Leghorn chickens housed in the same filtered air, positive pressure building, were inoculated at 1 day, 2, 4, 6, 8 and 10 weeks of age respectively, with a mixture of leukosis viruses of subgroups A and B. Birds which died during the experiment were examined for gross and microscopical lesions. The incidence of lymphoid leukosis (LL) in the various groups was inversely proportional to the age of first virus exposure, Le., mortality was 62.5% in the group inoculated at 1-day-old and decreased to zero in the group inoculated at 10 weeks of age. In the age group inoculated at 8 weeks, only one chicken succumbed to the disease. Congenital transmission of leukosis virus (LV) was detected only in chickens inoculated immediately after hatching or at 2 weeks of age. In the latter group, all leukosis virus positive embryos were derived from one hen only. A seventh age group, housed in a separate filtered air, positive pressure building, served as uninoculated controls. Challenge infection was performed on the same day with chickens of all groups at ages varying from 59 to 71 weeks. Testing of pooled embryo extracts collected between 1 and 15 weeks post challenge did not change the virus shedding pattern. Even the chickens of the control group which were challenged (for the first time) at 71 weeks of age remained negative for congenital transmission. The results of this experiment show that the chickens inoculated with LV at 4 weeks of age or older had a low incidence of LL (decreasing to zero when the chickens were inoculated at 10 weeks of age) but did not congenitally shed virus.     

Molecular and ultrastructural characterization of porcine hippurate-negative Brachyspira pilosicoli

Brachyspira pilosicoli, the causative agent of porcine intestinal spirochetosis, usually has hippurate-cleaving capacity. We have regularly isolated hippurate-negative B. pilosicoli from cases of porcine diarrhea. In this study, we show that these biochemically atypical B. pilosicoli isolates can be classified as B. pilosicoli. 16S ribosomal DNA was partially sequenced from eight hippurate-negative and two hippurate-positive B. pilosicoli-like isolates from seven herds. The differences in nucleotide sequence with B. pilosicoli P43/6/78 type strain were not associated with hippurate cleavage. In 877 bp, the hippurate-negative isolates had a similarity of 98.63 to 100% to the type strain, with the corresponding figures for the two hippurate-positive isolates being 98.86 and 100%. The nucleotide sequences of hippurate-positive isolates were identical to the respective sequences of hippurate-negative isolates from one herd. The DNA macrorestriction patterns of a total of 20 hippurate-negative and -positive B. pilosicoli isolates were diverse, and no clustering in conjunction with the hippurate reaction was found. In two herds, hippurate-positive and -negative B. pilosicoli isolates had a common macrorestriction pattern. The ultrastructure of hippurate-negative isolates was similar to the type strain. In conclusion, B. pilosicoli can be either hippurate positive or negative and, thus, the scheme for biochemical differentiation of porcine Brachyspira should be revised to include identification of hippurate-negative B. pilosicoli.     

Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens

One-week-old commercial layers were infected orally with 10⁸ colony forming units of Salmonella enterica serovar Enteritidis phage type 4. No mortality was observed. The inoculated organism was isolated in decreasing viable numbers from a number of tissues, particularly the spleen, liver and caeca. Organisms present in the spleen were primarily localized within macrophages. No Salmonella Enteritidis organisms were isolated between 10 and 24 weeks of age, when the experiment was terminated after several weeks of lay. When two groups of adult hens, housed with males, were infected, contaminated eggs were found within 2 weeks of infection in one of the experiments only. Progeny hatched from these eggs showed no mortality unless they were infected artificially with the S. Enteritidis strain. In this case, the percentage mortality fell as the hatches progressed, indicating increasing immunity to infection. The faecal excretion of the inoculated phage type 4 strain by infected but healthy progeny was followed. Although most birds ceased to excrete by 11 to 12 weeks of age, a small number of the birds continued to excrete until they themselves came into lay. The small numbers of birds in which this occurred indicates that tolerance to infection does not occur readily following infection of hens laying fertile eggs or in progeny birds infected before or within hours of hatching. Birds infected when they were less than 24 h old remained persistently infected until they were well into lay. However, control birds infected when 1 week old, on this occasion, showed a high level of excretion until the birds began to lay at 18 weeks. Inbred lines of chickens showing differences in their susceptibility to systemic salmonellosis did not show significant differences in the extent to which S. Enteritidis localized in the organs of the reproductive tract or in the number of infected eggs produced.     

Detection by PCR and isolation assays of the anaerobic intestinal spirochete Brachyspira aalborgi from the feces of captive nonhuman primates

The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the feces of captive nonhuman primates (n = 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the B. aalborgi PCR. B. aalborgi was not detected by PCR in any of the other animal or bird species tested, and B. pilosicoli was not detected in the primates or any of the other animals or birds. B. aalborgi was isolated from both PCR-positive vervet monkeys. This is the first time that B. aalborgi has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species.     

Studies on EDS-76 virus infection in laying chickens

A new strain (D61) of EDS-76 virus associated with an egg-drop syndrome in chickens was compared by neutralisation and haemagglutination-inhibition (HI) tests with two recognised strains (127, BC14). All three strains were serologically indistinguishable. Each strain lacked any demonstrable ability to lyse the red cells of chickens or ducks. To investigate its pathogenicity and immunogenicity, chickens of different ages were inoculated with the D61 strain per os. All birds produced eggs with aberrant shells in varying numbers, but internal quality was not affected. There were no other constitutional effects and no obvious clinical signs throughout. Fertility and hatchability remained unaffected. At 19 weeks, onset of laying was delayed by 9 days, and total daily egg production was eventually 15 to 20% lower than that of uninfected controls of the same age. The total daily egg production of older chickens was unaltered, except for approximately 2 weeks after infection at 26 weeks of age and for 5 weeks at 47 weeks of age. The highest number of eggs with abnormal shells was laid by chickens infected at 33 weeks (up to 38%) or 47 weeks of age (up to 40%). Infective virus was successfully recovered from the livers of chicks immediately after hatching from eggs laid by hens 7 to 11 days following infection. Neutralising, HI and precipitating antibodies developed within 6 days of infection, and were associated with the IgG class of immunoglobulins. Chicks hatched from eggs of infected hens possessed maternal antibodies with a half-life of 3 days; these antibodies conferred a passive immunity to challenge for approximately the first 4 weeks of life, based on the evidence of HI responses after challenge.     

Cyclosporin A, but not bursectomy, abolishes the protective immunity of chickens against Leucocytozoon caulleryi

Chickens that survive primary infection with Leucocytozoon caulleryi show strong resistance to reinfection. Using bursectomized (BX) or cyclosporin A (CsA)-treated chickens, we performed experiments to determine which type of immunity, humoral or cellular immunity, plays an important role in the resistance of chickens against reinfection with L. caulleryi. BX chickens were inoculated with 2, 20 or 200 sporozoites of L. caulleryi intravenously at 3 weeks of age. Some BX chickens which were inoculated with 2 or 20 sporozoites survived the primary infection. These birds had no parasites in their peripheral blood after challenge infection with 5 x 10(3) sporozoites, even though they had no antibody to L. caulleryi. In contrast, CsA-treated chickens had parasitemia, serum-soluble antigen and antibodies, as did untreated chickens during primary infection. After secondary infection, CsA-treated chickens had parasitemia and serum-soluble antigen, even though they had specific antibodies to L. caulleryi whereas untreated chickens showed no parasitemia. The number of CD4(+), CD8(+), T cell receptor (TCR) alpha ss-bearing cells and TCRgamma delta-bearing cells decreased markedly in the peripheral blood of CsA-treated chickens compared to those of untreated chickens. Lymphocyte proliferation in response to concanavalin A, and T cell growth factor production, were also markedly suppressed in CsA-treated chickens. These results suggest that cell-mediated immunity plays an important role in the development of resistance of chickens against reinfection with L. caulleryi.