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.

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.

[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.

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.

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.

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.

Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina.

We have investigated the expression of a calcium-binding protein, the S100 protein, in Eimeria brunetti and Eimeria acervulina stages. For this purpose, paraffin sections of distal ileum and bursa of Fabricius or duodenum from experimentally infected chickens were treated with anti-alpha-S100 (anti-alpha subunit of S100 protein) and anti-beta-S100 (anti-beta subunit of S100 protein) monoclonal antibodies and anti-S100 whole molecule polyclonal antibody. The avidin-biotin peroxidase method was used to demonstrate immunoreactivity. In the ileum, our results reveal a positive immunoreaction for the beta subunit and S100 whole molecule within the macrogametes of E. brunetti, whereas they were devoid of immunostaining after treatment of the paraffin sections with the anti-alpha-S100 antiserum. Schizonts and oocysts of E. brunetti and all the E. acervulina stages gave a negative reaction after treatment with any of the three antiserum used in the study. This result indicated that the S100 protein molecules within these stages were not recognized by the antibodies, suggesting that these molecules are different from those identified in macrogametes of E. brunetti. By contrast, in the epithelial cells, lining the lumen of the bursa of Fabricius, macrogametes of E. brunetti were stained by the three antibodies used. These results may indicate the existence of metabolic adaptations that enable the parasite to invade tissue sites different from those where the parasite usually develops.

Light-microscopic lesions associated with naturally occurring bursal cryptosporidiosis in chickens.

Information concerning histologic significance of naturally occurring bursal Cryptosporidium sp. infection in chickens was obtained by retrospective examination of histopathology reports and clinical data from histology accessions received during 1986. Cryptosporidiosis was diagnosed in 197 bursas. In two-thirds of the accessions, more than 50% of the examined bursas were infected with Cryptosporidium sp. The histologic morphologic lesion diagnosis for Cryptosporidium sp.-infected bursas most often was marked diffuse chronic-active superficial purulent protozoal bursitis with mucosal epithelial hyperplasia. Our study clearly indicates that Cryptosporidium sp. is associated with inflammation and disturbed growth in chicken bursas. Additionally, our data indicate that Cryptosporidium sp. infection is not dependent on bursal damage attributable to other agents, including infectious bursal disease virus.

Cryptosporidial infection in broiler chickens in Greece.

Trachea, bursa of Fabricius, and small intestine of broilers 5 to 50 days of age from 10 flocks with varying levels of morbidity and mortality were screened for the presence of Cryptosporidium spp. Cryptosporidial oocysts were found in 24.2% of the examined birds.

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.

Cryptosporidiosis in chickens from southern Spain.

Cryptosporidiosis in chickens from southern Spain is reported. Cryptosporidia were found in the trachea, esophagus, and epithelium of the bursa of Fabricius in chickens from different regions. In one flock, cryptosporidiosis was associated with respiratory problems and high mortality. In another flock, it was associated with low mortality and weight loss.

Eimeria brunetti in epithelial cells of the bursa of Fabricius in gnotobiotic chickens.

Germ-free and conventional chickens were infected with Lactobacillus acidophilus and/or Eimeria brunetti. Germ-free chickens acquiring single infections of E. brunetti were found to have lifecycle stages of the coccidial organism in the bursa of Fabricius.

Cryptosporidiosis of the bursa of Fabricius of chickens.

Light-microscope and electron-microscope studies of a coccidial organism found in the bursa of Fabricius from 3 chickens clearly established the parasite as belonging to the family Cryptosporiidae. Hyperplasia and heterophil infiltration were associated with the presence of organisms attached to the microvillus border of epithelial cells lining the plicae of the bursa of Fabricius. Although there were no clinical signs or gross lesions common to the 3 cases described, all had similar histologic lesions in the epithelium lining the bursa of Fabricius.

Demonstration of Eimeria tenella in bursa of Fabricius of chickens.

Coccidial life-cytle stages were detected in the bursa of Fabricius of broiler chickens inoculated with Eimeria tenella, whether or not the chickens had previously been infected with infectious bursal disease virus (IBDV). Chickens infected only with E. tenella had developing parasites in the lining epithelium, whereas chickens with both infections had gametocytes also in the epithelial cells surrounding numerous degenerating bursal cysts.

Intestinal and bursal cryptosporidiosis in turkeys following inoculation with Cryptosporidium sp. isolated from commercial poults.

Cryptosporidium meleagridis oocysts, originally isolated from droppings of commercial turkey poults with increased mortality due to viral (reovirus) hepatitis and enteritis, were treated with peracetic acid to kill companion bacteria and viruses and then propagated by passage in young turkeys. Thirty-eight 5-day-old large white turkey poults were inoculated by crop gavage with 500,000 cryptosporidial oocysts and compared with 40 uninoculated poults. Cryptosporidial oocysts shedding began 3 days postinoculation (PI), peaked on day 4 PI, and persisted at a low level for the duration of the 21-day trial. Low to moderate cryptosporidial infections of the ileal mucosa (days 3, 6, and 15 PI), cecal mucosa (days 3, 6, and 21 PI), and bursa of Fabricius (days 6, 12, 15 and 21 PI) were found on histopathological examination. There were no differences in mean body weights between the inoculated and uninoculated groups, and no mortality or clinical signs of disease were seen in either group.

The relationship of Cryptosporidium sp. infection of the bursa of Fabricius, intestinal tract, and respiratory system of chickens in Georgia, 1974-1988.

During 1974-1984, cryptosporidiosis was diagnosed in slightly more than 1% (63/6050 = 1.04%) of histopathology reports on chickens and turkeys made at two Georgia diagnostic laboratories. During 1985-1988, cryptosporidiosis was diagnosed in almost 6% (157/2622 = 5.99%) of histopathology reports on chickens and turkeys made at the two laboratories. Intestinal and respiratory Cryptosporidium sp. infections have increased significantly (p less than 0.01) with time. During the same period, there was no statistical pattern for bursa of Fabricius Cryptosporidium sp. infections, and there was no relationship between the incidence of bursal infections and the incidence of either respiratory or intestinal infections.