Forests of opportunities and mischief: disentangling the interactions between forests, parasites and immune responses.

Habitat characteristics determine the presence of individuals through resource availability, but at the same time, such features also influence the occurrence of parasites. We analyzed how birds respond to changes in interior forest structures, to forest management regimes, and to the risk of haemosporidian infections. We captured and took blood samples from blackcaps (Sylvia atricapilla) and chaffinches (Fringilla coelebs) in three different forest types (beech, mixed deciduous, spruce). We measured birds' body asymmetries, detected avian haemosporidians, and counted white blood cells as an immune measure of each individual per forest type. We used, to our knowledge for the first time, continuous forest structural parameters to quantify habitat structure, and found significant effects of habitat structure on parasite prevalence that previously have been undetected. We found three times higher prevalence for blackcaps compared with chaffinches. Parasite intensity varied significantly within host species depending on forest type, being lowest in beech forests for both host species. Structurally complex habitats with a high degree of entropy had a positive effect on the likelihood of acquiring an infection, but the effect on prevalence was negative for forest sections with a south facing aspect. For blackcaps, forest gaps also had a positive effect on prevalence, but canopy height had a negative one. Our results suggest that forest types and variations in forest structure influence the likelihood of acquiring an infection, which subsequently has an influence on host health status and body condition; however, responses to some environmental factors are host-specific.

The Gametocytes of Leucocytozoon sabrazesi Infect Chicken Thrombocytes, Not Other Blood Cells.

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission.

A blurring of life-history lines: Immune function, molt and reproduction in a highly stable environment.

Rufous-collared sparrows (Zonotrichia capensis peruviensis) from valleys in the Atacama Desert of Chile, live in an extremely stable environment, and exhibit overlap in molt and reproduction, with valley-specific differences in the proportion of birds engaged in both. To better understand the mechanistic pathways underlying the timing of life-history transitions, we examined the relationships among baseline and stress-induced levels of corticosterone (CORT), testosterone, and bacteria-killing ability of the blood plasma (BKA), as well as haemosporidian parasite infections and the genetic structure of two groups of sparrows from separate valleys over the course of a year. Birds neither molting nor breeding had the lowest BKA, but there were no differences among the other three categories of molt-reproductive stage. BKA varied over the year, with birds in May/June exhibiting significantly lower levels of BKA than the rest of the year. We also documented differences in the direction of the relationship between CORT and BKA at different times during the year. The direction of these relationships coincides with some trends in molt and reproductive stage, but differs enough to indicate that these birds exhibit individual-level plasticity, or population-level variability, in coordinating hypothalamo-pituitary-adrenal axis activity with life-history stage. We found weak preliminary evidence for genetic differentiation between the two populations, but not enough to indicate genetic isolation. No birds were infected with haemosporidia, which may be indicative of reduced parasite pressure in deserts. The data suggest that these birds may not trade off among different life-history components, but rather are able to invest in multiple life-history components based on their condition.

Antibody response and protective immunity of chickens vaccinated with booster dose of recombinant oil-adjuvanted Leucocytozoon caulleryi subunit vaccine.

Leucocytozoon caulleryi is an economically important poultry pathogen that causes subclinical to fatal disease in chickens. Because of limited preventive and treatment options against this disease, an oil-adjuvanted recombinant vaccine (O-rR7) targeting the R7 protein of L. caulleryi second-generation schizonts was developed. Different vaccination programs, namely, single vaccination at 45 days (0.1-ml dose), single vaccination at 130 days (0.25 ml), and initial vaccination at 45 days (0.1 ml) followed by a booster dose at 130 days (0.25 ml) were explored to compare the effects of single and booster vaccination on antibody response, duration of protective immunity, and degree of clinical signs after experimental L. caulleryi infection. Of the three treatments groups, initial vaccination at 45 days followed by a booster vaccination at 130 days of age resulted to rapid increase in antibody titers, which persisted for up to 182 days. Antibody titers reached peak values 35 days and 14 days after initial and booster vaccination, respectively. In comparison, single vaccination at 45 days of age resulted in production of antibodies above 1600 ELISA units for 56 days postvaccination, and single vaccination at 130 days of age produced peak antibody titers 35 days postvaccination, which remained above 1600 ELISA units for 126 days. Experimental infection of L. caulleryi at 256 days, when antibody titers had waned, did not result to severe clinical disease in chickens that received booster vaccination, whereas mild to severe disease was observed in chickens that received a single vaccination. Evaluation of immune response at 15 and 21 days postinfection showed that chickens that received booster vaccination had a twofold increase (P < 0.01) in antibody titers as compared to those receiving a single vaccination. Administering booster shots of O-rR7 is therefore recommended, especially in farms located in areas where Leucocytozoon is endemic.

Blood parasites mediate morph-specific maintenance costs in a colour polymorphic wild bird.

Parasites can mediate profound negative effects on host fitness. Colour polymorphism has been suggested to covary genetically with intrinsic physiological properties. Tawny owl colour polymorphism is highly heritable with two main morphs, grey and brown. We show that experimental medication acts to reduce blood parasites and that medicated grey females maintain body mass during breeding, whereas medicated brown females decline in body mass similar to control females of both morphs. We find no effect of medication on general immunoglobulin levels, antigen-specific humoral response or H/L ratio. In the descriptive data, both morphs have similar blood parasite infection rates, but blood parasite infection is associated with decreased body mass in brown but not in grey females. We conclude that blood parasite infection primarily has somatic costs, which differ between the two highly heritable tawny owl colour morphs with more pronounced costs in the grey (little pigmented) morph than in the brown (heavily pigmented) morph. Because our descriptive results imply the opposite pattern, our findings highlight the need of experimental manipulation when studying heritable variation in hosts' response to parasitism.

A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen.

A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

Increase of antibody titer against Leucocytozoon caulleryi by oral administration of recombinant R7 antigen.

In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.

Field efficacy of recombinant R7 vaccine against chicken leucocytozoonosis.

Effectiveness of vaccine that used recombinant R7 protein (rR7) as antigen that is derived from second-generation schizont (2GS) of Leucocytozoon caulleryi was verified under a field condition against chicken leucocytozoonosis. Chickens reared in a poultry farm where the chickens are attacked by leucocytozoonosis in every year were inoculated with oil-adjuvanted rR7 vaccine (O-rR7), and the immunized chickens were found to have production of antibodies against 2GS at a high level by one shot. Leucocytozoonosis was observed at post-injection. During the epidemic period of leucocytozoonosis, the unique clinical signs of the disease such as discharge of green feces and anemia, and also parasitemia were observed, however, compared to chickens in control group, those in O-rR7 vaccinated group had significantly slight symptoms (P<0.05). In addition to this, immunized chickens had better result of egg production than the unvaccinated chickens did, and the maximum difference of egg production rate, 22%, was observed at the peak of the disease. In conclusion, it is verified that O-rR7 vaccine has efficacy against leucocytozoonosis under field condition, and this vaccine can be put into practical use.

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.

Characterization of monoclonal antibodies against the second-generation schizonts of Leucocytozoon caulleryi.

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.

Immunoblot analysis of humoral immune responses to Leucocytozoon caulleryi in chickens.

The humoral antibody responses of chickens infected or immunized with Leucocytozoon caulleryi were analyzed by immunoblot comparing with protection against challenge infection, agar-gel precipitation (AGP) test, enzyme-linked immunosorbent assay (ELISA), and parasitemia. IgG antibodies in the sera from chickens infected with L. caulleryi were found to react with 20-35 bands of approximate molecular weights 25-300 kDa, such as 33, 44, 58, 79, 94, and 141 kDa from 3 to 50 wk after infection. In chickens immunized with schizont antigens from L. caulleryi, several bands were reacted with sera 2 wk after the second immunization, e.g., 36, 58, 71, 81, 97, 112, and 123 kDa. Chickens that recovered from the primary infection showed complete protection against reinfection, whereas immunized chickens showed partial protection against challenge infection. These results suggest that the difference in antibody response to schizont antigen might cause the difference in protection between immunized and infected chickens.

Immunogenicity of Leucocytozoon caulleryi sporozoites and their reactivity with specific immune sera.

The immunogenicity of Leucocytozoon caulleryi sporozoites for chickens and their reactivity in vitro with specific immune sera were studied. Almost all of the chickens that had been immunized with the sporozoite antigens survived the sporozoite challenge. The degree of parasitemia observed in the immunized chickens was significantly lower than that found in the nonimmunized chickens. Specific antibodies against sporozoites were tested by the circumsporozoite precipitation (CSP) reaction. Antibodies were demonstrated in the sera of chickens that had been immunized with the sporozoite antigens or chickens that had recovered from a primary infection with L. caulleryi sporozoites. When viable mature sporozoites were incubated in vitro with serum from immune chickens, agglutination and a long, thread-like precipitate at one end of the sporozoite could be seen within a few minutes under a phase-contrast microscope. The effects of specific immune serum on the infectivity of sporozoites were examined by the sporozoite neutralization activity (SNA) test. Sporozoites that had been incubated in vitro with serum from immune chickens lost their infectivity to chickens. The CSP reaction and the SNA test in L. caulleryi infection were stage- and species-specific.

Extraction of Haemoproteus columbae (Haemosporina: Haemoproteidae) antigen from rock dove pigeons (Columba livia) and its use in an antibody ELISA.

Erythrocytic stages of Haemoproteus columbae were extracted from the cytoplasm of nucleated red blood cells (RBC) of Rock dove pigeons (Columba livia) using cationic detergent (N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane [EDTA-20]) and discontinuous Percoll gradient density. Crude RBC extract (CRBCE) antigen was prepared. Parasitized RBCs were more resistant to EDTA-20 action than unparasitized cells. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of anti-H. columbae immunoglobulins in 30 wild-captured C. livia. Whole blood, serum, and dried blood on filter paper gave similar results; the latter was selected for sampling convenience. Optimal antigen concentration was 5 micrograms/ml, and anti-H. columbae immunoglobulins were detectable at a 10(-4.11) dilutions. The binding efficacy of anti-chicken IgG to the pigeon immunoglobulins was significantly higher than anti-duck IgG or anti-turkey IgG. Parasitemia by Giemsa-stained thin blood smears ranged from 20.0 to 47.5%, mean = 32.4 +/- 8.3%; 17 of 30 birds had multiply infected RBCs with a mean parasitemia of 2.4 +/- 1.1%, range 0.7-4.9%; 27 birds were positive by the ELISA. No clinical signs of infection were observed. ELISA absorbance values were not correlated with the level of parasitemia in individual birds. All pigeons were negative for anti-Plasmodium relictum and anti-P. elongatum immunoglobulins as determined by ELISA. The pigeons were not subclinically infected with Plasmodium spp. as determined by inoculation of domestic ducklings with blood from dexamethasone-immunosuppressed pigeons.

Protection against chicken leucocytozoonosis provided by immunization with spleen homogenate infected with Leucocytozoon caulleryi.

Protection against chicken leucocytozoonosis was assessed in chickens immunized with spleen homogenates from chickens that had received sporozoites of Leucocytozoon caulleryi 7 or 13 days previously. Chickens immunized with the homogenate were challenged with sporozoites of L. caulleryi and observed for changes in clinical signs, parasitemia, serum-soluble antigen, and antibody responses. In chickens immunized with either the 7-day or 13-day homogenate, clinical signs and parasitemia were moderate, mild or absent. This was the case both after immunization with the homogenate and after sporozoite challenge. Immunization with spleen homogenates demonstrated protection against chicken leucocytozoonosis.