Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.

BACKGROUND: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Post mortem findings and their relation to AA amyloidosis in free-ranging Herring gulls (Larus argentatus).

Since the late 1990s, high mortality and declining populations have been reported among sea birds including Herring gulls (Larus argentatus) from the Baltic Sea area in Northern Europe. Repeated BoNT type C/D botulism outbreaks have occurred, but it remains unclear whether this is the sole and primary cause of mortality. Thiamine deficiency has also been suggested as a causal or contributing factor. With this study, we aimed to investigate gross and microscopic pathology in Herring gulls from affected breeding sites in Sweden in search of contributing diseases. Herring gulls from Iceland served as controls. Necropsies and histopathology were performed on 75 birds, of which 12 showed signs of disease at the time of necropsy. Parasites of various classes and tissues were commonly observed independent of host age, e.g. oesophageal capillariosis and nematode infection in the proventriculus and gizzard with severe inflammation, air sac larid pentastomes and bursal trematodiasis in pre-fledglings. Gross and microscopic findings are described. Notably, amyloidosis was diagnosed in 93 and 33% of the adult birds from Sweden and Iceland, respectively (p<0.001), with more pronounced deposits in Swedish birds (p<0.001). Gastrointestinal deposits were observed in the walls of arteries or arterioles, and occasionally in villi near the mucosal surface. Amyloid was identified within the intestinal lumen in one severely affected gull suggesting the possibility of oral seeding and the existence of a primed state as previously described in some mammals and chickens. This could speculatively explain the high occurrence and previously reported rapid onset of amyloidosis upon inflammation or captivity in Herring gulls. Amyloid-induced malabsorbtion is also a possibility. The Herring gull SAA/AA protein sequence was shown to be highly conserved but differed at the N-terminus from other avian species.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella.

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

Effects of different inoculation routes on the parasitic sites of Cryptosporidium baileyi infection in chickens.

Cryptosporidiosis is prevalent in domesticated, caged, and wild birds. Cryptosporidium baileyi, an ascendant species of avian Cryptosporidium, is an important pathogen. It causes respiratory disease in chickens, especially chickens younger than 50 days. In this study, SEM, histological, semi-quantitative PCR, and nested PCR techniques were used to explore the impact of different inoculation routes on sites of C. baileyi infection in chickens. Results showed that inoculation with sporozoites or oocysts via the rectum was an effective means of causing infection. This may provide an important reference for the development of the transfection system of C. baileyi in chickens. Numerous endogenous stages of C. baileyi were observed in the bursas of Fabricius (BF) and cloacas of chickens inoculated with sporozoites or oocysts via the rectum, but no parasite was seen in the tracheas of any of these chickens. In chickens infected with oocysts via the crop, the number of parasites in the BF was approximately 23-fold more than in the trachea. All blood samples collected after inoculation were negative for C. baileyi. These data show that C. baileyi was not transferred by blood circulation between the BF and respiratory tract. Different routes of inoculation were here found to distinctly affect sites of parasitism in chickens. These findings may facilitate further understanding of the biology of C. baileyi and efforts to control avian cryptosporidiosis.

Cryptosporidium baileyi--infection in Red-breasted Merganser (Mergus serrator) ducklings from a zoological garden.

Infection of the Bursa of Fabricius with Cryptosporidium baileyi was diagnosed in a group of hand reared Red-breasted Merganser (Mergus serrator) ducklings during one breeding season in a German zoological garden.The birds had died in an emaciated and anaemic state after problems with spontaneous feeding. The bursae were infected with moderate to high numbers of cryptosporidia, which were associated with hyperplasia, degeneration and sloughing of the affected epithelial cells and mild heterophilic bursitis, lesions typically seen in bursal cryptosporidiosis in other avian species. In addition, lymphatic tissue was nearly absent, which was probably caused by chronic stress and malnutrition related to the rearing of these highly stress-sensitive birds in an artificial environment. Companion ducklings from stress-resistant, spontaneously feeding species from the zoological collection were used to calm the Mergansers, but may have introduced the cryptosporidia into the rearing boxes. Another possible source for the introduction of C. baileyi were adult mallards (Anas platyrhynchos), which were used to increase the hatching rate by a phase of natural breeding in the middle third of the incubation period of the Merganser eggs.

[Morpho-functional changes of cloacal bursa of the herring gull in experimental infection with gull tapeworm].

Using the methods of light microscopy, the quantitative changes in the cells of the cloacal bursa of herring gull (Larus argentatus mongolicus) chicks were studied during experimental infection with gull-tapeworm Diphyllobothrium dendriticum. The area of the follicles within the bursa and the number of eosinophils in the infected chicks were found to increase. In the follicles the number of blast cells and large lymphocytes was elevated. The reduction of small lymphocytes and plasma cells numbers in the infected gulls may be due to a slower cell cycle or cell migration into the inflammatory focus. Overall, the changes in the bursa indicate the suppression of the B-link of the host immune system in tapeworm invasion.

[The reaction of the bursa and thymus eosinophils in the herring gull after the experimental infection with gull-tapeworm].

Using the methods of light microscopy, eosinophil topography, quantitative and qualitative changes (degranulation level of and a cationic protein content) were studied in the thymus and bursa of 36 herring gull nestlings Larus argentatus mongolicus (Suskin, 1925) 2 weeks after experimental infestation with gull-tapeworm Diphyllobothrium dendriticum (Nitsch, 1824). Eosinophils in the thymus were located in trabecules, mainly close to the blood vessels, thymic (Hassall's) corpuscles and also directly inside them, while in the bursa they were found within the internodular space. As compared with the control bird counts, relative eosinophil count in the birds with an average invasion intensity was increased 3.8 times in the thymus and 2.5 times in the bursa. In birds with high invasion intensity, these counts were increased 4 times in the thymus and 1.2 times in the bursa.

Experimental study on respiratory cryptosprodiosis.

Cryptosporidiosis has a worldwide geographical and zoological distribution. Cryptosporidium baileyi oocysts were isolated from the bursa of Fabricius (BF) of 4 weeks-old of 100 broilers. Twenty five broiler chicks of 2 day old free from cryptosporidial infection were inoculated intratracheally at a dose of 1 x 10(6) chicks. Five chicks were sacrificed at 4th, 8th, 12th, 16th, and 20th day postinoculation. Selected tissues from bursa of Fabricius, cloaca, colon, caeci and tracheas were taken and examined directly by scraping technique for endogenous stages. The distribution of C. baileyi in different tissues in the case of intratracheal inoculation; trachea 23 (92 %) chicks infected, bursa of Fabricius 21 (84%) chicks, Cloaca 5 (16 %) chicks, Colon 1 (4%) chick and the caeci showing no infection. Daily examination of the intratracheal droppings inoculation was done at the 5th day and lasted for 18-20 days, the patent period.

Histomonas meleagridis: immunohistochemical localization of parasitic cells in formalin-fixed, paraffin-embedded tissue sections of experimentally infected turkeys demonstrates the wide spread of the parasite in its host.

In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas meleagridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific for histomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae and Blastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity of the technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n=5) of caecum, liver, spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each of proventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected in blood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative study with routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical technique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain new insights into the mechanisms of interaction with the host tissues.

Experimental coccidiosis provoked by Eimeria acervulina in chicks simultaneously fed on ochratoxin A contaminated diet.

The progression of coccidiosis provoked by Eimeria acervulina was followed in chicks fed on OTA-contaminated as well as on OTA-free diets. More heavy progress of duodenal coccidiosis, including mortality, occurred in OTA-treated chicks as can be seen from the higher value of lesion (3.50) and oocyst (31.65) indices. A stronger decrease of serum total protein was found in OTA-treated chicks (22.80 g/l) than in chicks infected with E. acervulina(24.20 g/l), but that decrease was strongest in chicks treated with OTA and simultaneously infected with E. acervulina (19.71 g/l). The serum concentration of uric acid was significantly increased in all chicks exposed to OTA, most notably in those additionally infected with E. acervulina (1020.6 (micro mol/L), whereas the serum enzyme activity of AST was increased only in chicks infected with E. acervulina and highest in those fed OTA contaminated diet (122.2 U/L). OTA induced degenerative changes in kidneys, liver and heart as well as a depletion of lymphoid tissue in the lymphoid organs and a decrease of body weight. Coccidiosis induced only a slight growth depression and duodenal hemorrhages in addition to characteristic duodenal damages. The impairment of kidney function, histopathological changes and general growth depression were stronger when chicks infected with E. acervulina were also given OTA.

A recombinant attenuated Salmonella enterica serovar Typhimurium vaccine encoding Eimeria acervulina antigen offers protection against E. acervulina challenge.

Coccidiosis is a ubiquitous disease caused by intestinal protozoan parasites belonging to several distinct species of the genus Eimeria. Cell-mediated immunity (CMI) is critically important for protection against Eimeria; thus, our approach utilizes the bacterial type III secretion system (TTSS) to deliver an antigen directly into the cell cytoplasm of the immunized host and into the major histocompatibility complex class I antigen-processing pathway for induction of CMI and antigen-specific cytotoxic T-lymphocyte responses in particular. To accomplish this goal, Eimeria genes encoding the sporozoite antigen EASZ240 and the merozoite antigen EAMZ250 were fused to the Salmonella enterica serovar Typhimurium effector protein gene sptP in the parental pYA3653 vector, yielding pYA3657 and pYA3658, respectively. SptP protein is secreted by the TTSS of Salmonella and translocated into the cytoplasm of immunized host cells. The host strain chromosomal copy of the sptP gene was deleted and replaced by a reporter gene, xylE. The newly constructed vectors pYA3657 and pYA3658 were introduced into host strain chi8879 (DeltaphoP233 DeltasptP1033::xylEDelta asdA16). This strain is an attenuated derivative of the highly virulent strain UK-1. When strain chi8879(pYA3653) as the vector control and strain chi8879 harboring pYA3657 or pYA3658 were used to orally immunize day-of-hatch chicks, colonization of the bursa, spleen, and liver was observed, with peak titers 6 to 9 days postimmunization. In vitro experiments show that the EASZ240 antigen is secreted into the culture supernatant via the TTSS and that it is delivered into the cytoplasm of Int-407 cells by the TTSS. In vivo experiments indicate that both humoral and cell-mediated immune responses are induced in chickens vaccinated with a recombinant attenuated Salmonella serovar Typhimurium vaccine, which leads to significant protection against Eimeria challenge.

Urotocus rossittensis (Trematoda: Digenea: Leucochloriidae) in the scarlet-rumped tanager, Ramphocelus passerinii, and common bush tanager, Chlorospingus ophthalmicus (Passeriformes: Thraupidae), from the Area de Conservación Guanacaste, Costa Rica, with taxonomic revision of the genus and revised key to the leuchochloridiid-like Brachylaimoidea.

Urotocus rossittensis occurs in the bursa Fabricii of the scarlet-rumped tanager, Ramphocelus passerinii, and the common bush tanager, Chlorospingus ophthalmicus, from the Area de Conservación Guanacaste, Costa Rica. Morphological examination of type material of U. fusiformis and U. kenyensis and Costa Rican specimens suggests that U. fusiformis and U. kenyensis are indistinguishable from U. rossittensis. Confirmed accounts of Urotocus spp. refer to a single adult morphotype whose geographic distribution includes the Palearctic, Africa, Nearctic, and northern Neotropics.

Cryptosporidium infection in domestic geese (Anser anser f. domestica) detected by in-situ hybridization.

An in-situ hybridization (ISH) procedure was developed for the detection of Cryptosporidium sp. in paraffin wax-embedded tissues with a digoxigenin-labelled probe targeting the 18S rRNA. This technique was used in addition to traditional methods, such as haematoxylin and eosin staining, periodic acid-Schiff reaction, transmission electron microscopy and the polymerase chain reaction, to examine the bursa of Fabricius (BF), conjunctiva and other tissues from 20 domestic geese aged 16-36 days for the presence of cryptosporidia. Positive signals were found to a moderate or marked extent in both conjunctival samples (89%) and BF samples (88%) but not in other tissues. Sequencing of the PCR amplification product revealed identity with Cryptosporidium baileyi. The infected geese showed no clinical signs and only scanty histological lesions. These results confirm reports showing that young waterfowl are especially vulnerable to cryptosporidium infection and indicate that the BF and conjunctiva are the preferred sites for the presence of the protozoon. ISH proved a good method for detecting and identifying even small numbers of cryptosporidia in tissue sections.

[Distribution of trematodes of the family Prosthogonimidae in river and lake ecological systems in the south of the Western Siberia].

The results of long-term investigations (1994-2003) of an infection rate of trematodes of the family Prosthogonimidae in the first intermediate hosts (snails of the family Bithyniidae) and in the final hosts (birds) from a basin of the Chany Lake (Western Siberia) are discussed. A total of 1824 specimens of Bithynia tentaculata (L., 1758) and 7166 specimens of Opisthorchophorus troscheli (Paasch, 1842) have been collected from eight rivers and two lakes in the south of the Western Siberia. Data of incomplete helminthological dissection of 225 young water-fowl of 18 species were analyzed. Birds were obtained in July, August and September, 1996-2003 from the Chany lake basin. Maritas of the trematode family Prosthogonimidae were revealed in bursa Fabricii (26.12%) of seven bird species of two orders (Anseriformes and Gruiformes): Grey Lag-Goose Anser anser (L.) (Infection rate-28.6% +/- 12.1), Mallard Anas platyrhynchos L. (27.5% +/- 6.2), Shoveler--A. clypeata L. (22.2% +/- 13.9), Pochard Aythya ferina (L.) (51.7% +/- 9.3), Coot Fulica atra L. (28.6% +/- 5.6), Tufted Duck A. fuligula (L.) (1 case), Wigeon A. penelope L. (1 case). Average intensity of invasion is 5.8 marita specimens per infected bird. The maximal number of parasites (37 maritas), and also the highest average intensity of invasion are recorded for the Coot. The 342 maritas of the family Prosthogonimidae belong to three species: Prosthogonimus ovatus (Rud., 1803) (49.7%), P. cuneatus (Rud., 1809) (32.8%) and Schistogonimus rarus (Braun, 1901) (14.6%); these trematode species have been recorded in 35, 12 and 8 bird species, respectively. Grey Lag-Goose, Shoveler, Pochard and Tufted Duck are new host records for P. cuneatus in for Western Siberia. Parthenitae of trematodes (Prosthogonimidae) were revealed from eight populations of bithyniid snails. The infection rate of the trematodes (parthenitae and cercariae) in population of bithyniid snails from lake ecological system varied 6.55-54.4%, and in river ecological systems in was 0.6-4.2 %. The infection rate of trematodes (parthenitae and cercariae) of the first intermediate hosts was 2.64% that is significantly lower than that of the final hosts from the Chany Lake basin. Both O. troscheli and B. tentaculata were noted as the first intermediate hosts of prosthogonimid trematodes of three species: P. ovatus, P. cuneatus and S. rarus. All three species of trematode were found in three water reservoirs: Ob River (B. tentaculata), Kargat River (O. troscheli) and Chany Lake (O. troscheli). Thus it is found out that 39 bird species can be the final hosts of prosthogonimid trematodes. About a quarter of nestlings is infected with these trematodes. The infection rate of parthenitae in snails from the lake ecosystems is significantly higher than in the river ecosystems.

Histomoniasis in the bursa of fabricius of chickens.

Histomoniasis was diagnosed in a flock of 6-wk-old commercial chickens. Clinical signs included depression, stilted gait, inappetence, and a slight increase in mortality. At necropsy, there were pale-yellow to dark-gray circular and depressed necrotic lesions in the liver. The ceca were enlarged and impacted with caseous cores. Cecal worms were not observed either at necropsy or on histopathology. Histomonads were demonstrated microscopically within the bursa of Fabricius in addition to the liver, ceca, and spleen. This is the first report of the presence of histomonads in the bursa of Fabricius in commercial chickens.

Viability of preserved Cryptosporidium baileyi oocysts.

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10(7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.

Occurrence of intermediate hosts and structure of digenean communities of the black-headed gull, Larus ridibundus (L.).

The factors contributing to the similarity between digenean parasite assemblages parasitizing the black-headed gull were investigated. Thirteen different digenean species from the digestive tract and the bursa of Fabricus were found. We predicted that the structure of parasite communities is not random at either component community or infracommunity levels. We searched for nested patterns in the digenean community, and explored the possible factors contributing to nestedness. We found that digenean species which occupy a narrow range of intermediate hosts are placed out of order in the nested matrix. The influence of several variables related to the water reservoir, geographical distances, and the abundance of intermediate hosts on the species diversity and similarity of parasite communities were tested as well. Because of the complexity in bird digenean life-cycles we supposed that intermediate hosts and ecological characters of their environment could play a major role. We showed that the presence of intermediate hosts is the factor limiting the qualitative and quantitative similarity of parasite communities among different host populations as well as influencing the digenean species diversity in the definitive host. The similarity in abundance of intermediate hosts between different localities was facilitated by the presence of vegetation in water reservoir and more diversified type of water bottom. Digenean species diversity was higher when the water reservoir was exposed to temporal drying. We hypothesized that this factor could facilitate the inclusion of molluscs infected by digeneans in the diet of birds. Both species diversity and dominance were influenced by the bottom type of water reservoir and temporal drying up. Geographical distances between localities influenced the quantitative similarity of digenean communities as well as species richness.

Viability of preserved Cryptosporidium baileyi oocysts

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10 (7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.