Verification of immunosuppression in chicks caused by Cryptosporidium baileyi infection using Brucella abortus strain 1119-3

Cryptosporidium baileyi appears to be present wherever avian hosts are raised commercially. It is a primary pathogen that can produce respiratory and/or intestinal disease resulting in morbidity and mortality and generally invades the epithelium of the cloaca and the bursa of Fabricius, the essential organ for the development of humoral immunocompetence in young chicks (Claflin et al., 1966; Cooper et al., 1966).

In a series of studies to document the immunosuppressive effect caused by the protozoan infection on humoral immunity to other poultry infections, we have shown previously that infection with C. baileyi in early life stage depressed the ability of the chicks to respond to sRBC (Rhee et al., 1998b). Moreover, recent investigations have also demonstrated that 2-day-old chicks initially infected with the protozoon showed a decreasing tendency of the immune responses following vaccination against Newcastle disease (ND) virus and avian infectious bronchitis (IB) virus, respectively (Rhee et al., 1998a & 1998c). Such immunosuppression to sRBC, ND virus and IB virus in chicks appears to be attributed to a marked diffuse chronic superficial purulent bursitis caused by C. baileyi (Rhee et al., 1997). Taken together, it is suggested that cryptosporidiosis could predispose chicks to secondary invasion by other important pathogens. Therefore, the present study examined this possibility using B. abortus strain 1119-3 as a corroborative indicator in C. baileyi infected chicks to verify the status of immunosuppression.

Oocysts used in the present study have been maintained in our laboratory since 1990, as previously described by Rhee et al. (1997). Bacteria were kindly provided by the Department of Veterinary Public Health, Chonbuk National University and cultured in Brucella agar and Brucella broth (Difco Laboratories, Detroit, USA) at 37℃ using 10% CO₂ in descending order. Two-day-old SPF chicks (Dekalb-Warren, Sex-Sal-Link, male) were divided into three experimental groups; infected (infected with both C. baileyi and B. abortus), uninfected (infected with B. abortus, but not with C. baileyi) and intrinsic control groups (negative control: uninfected with both C. baileyi and B. abortus). Chicks were orally inoculated each with a single dose of 2×10⁶ oocysts of C. baileyi, and 15 chicks in each group were injected intramuscularly each with 0.3 ml of B. abortus strain 1119-3 containing 1×10⁹ living organisms on 14 days after C. baileyi infection. Procedures used for the feeding of chicks and the examination of fecal samples were referred to those described previously (Rhee et al., 1997).

The serum samples were collected from each bird at an interval of 3-6 days over a period of 36 days postinoculation (PI) with the bacteria and the serum agglutination titers to B. abortus were estimated by buffered plate agglutination test (PAT) using a commercially available antigen prepared by B. abortus strain 1119-3 for PAT, as recommended by the manufacturer (Dae Sung Microbial Laboratories, Seoul, Korea). To prepare the antigen, B. abortus strain 1119-3 concentrate was diluted to a final concentration of 0.5% phenol (0.5 g phenol in 100 ml of normal saline solution) along with crystal violet and brilliant green. Thus doubling dilutions of serum from 1.25 to 80 µl were transferred to 10 well glass plates marked in 1.5×1.5 cm, respectively. Aliquots (30 µl) of the antigen were added to appropriate wells of the previously prepared plates. The plates were rotated three times in a tilting motion, and then incubated for 4 min in a humid chamber at an ambient room temperature (20-25℃). They were then incubated again after three times rotation as described above. At this point, the plates were removed, rotated and observed for visible agglutination.

The test in each isolator was repeated at least three times with similar results. PAT levels to B. abortus between infected and uninfected groups were analyzed by Student's t-test.

The results of the serum agglutination titers to B. abortus strain 1119-3 between C. baileyi-infected and uninfected chicks were summarized in Table 1. The titers were not detected at any time in the samples from 15 intrinsic control chicks, indicating that the titers in both infected and uninfected chicks had developed specific antibody in response to the bacterial infection and that the bacteria did not spread to the intrinsic control chicks. The titers were not observed at day 3 PI in samples from both infected and uninfected chicks.

Table 1.

Fluctuations of log₂ based serum agglutination titers to Brucella abortus in Cryptosporidium baileyi inoculated chicks

The serological immune response to the bacterial infection of chicks which were previously infected with the protozoon was significantly lower than that of uninfected chicks throughout the experiment (P<0.05), never reaching a mean greater than log₂ based titer of 2.07 without an evident peak. The difference in peak titers between infected and uninfected chicks at 15 days PI was exceedingly significant (P=0.0001). It is supposed that C. baileyi infection caused a disturbance of the ontogeny of B-cell by hyperplasia and thickening of bursal epithelium and a mild to moderate depletion of lymphocytes in bursal follicles (Rhee et al., 1997).

Since the humoral immune response to ^{B. abortus} was observed to assess the effect of surgical and chemical bursectomy (Claflin et al., 1966; Cooper et al., 1966), a number of researchers have used this bacterium to monitor the antibody response in chicks (Hopkins et al., 1979; van der Zijpp et el., 1986; Kreukniet et al., 1992). Although the serological response to the bacteria is not high in chicks, aside from ND and IB, the current work showed that measurement of the immune response to B. abortus is a suitable, satisfactory method for assessing immunosuppression caused by cryptosporidiosis. In addition, the result suggests that the serum agglutination test may be applicable for the measurement of immunosuppression in field situations where it would not be possible to use other avian pathogens because of interferences of maternal antibody or natural infection to the test stimulant. Moreover, the serum agglutination test may have simple, practical advantages over the hemagglutination inhibition test in detection of ND and IB antibodies.

In the infected chicks, oocyst output in the excrements was presumed to show similar profiles to those of our previous study (Rhee et al., 1998b). However, oocysts were not detected in the excremental samples of the intrinsic control and the uninfected chicks during this period.

Although infectious bursal disease had a significant effect on response to ND virus vaccination (Faragher et al., 1974), it is corroborate that cryptosporidiosis leads to a significant reduction in the antibody response after vaccination with ND virus and IB virus based on the results from the present and previous studies (Rhee et al., 1998a & 1998c). Taken together, the present study highlights immunosuppression attributed by cryptosporidiosis in early life stage of chicks, which is presumably the first consideration worldwidely. Therefore, we propose that cryptosporidiosis should be eradicated preferentially by proper sanitation and immunization to prevent important infectious diseases in poultry industry, even though there are no proven recommended programs. Finally, further work is required to determine whether the degree of immunosuppression could have a practical effect upon the severity of other avian disease.