Trypanosoma, Gruby

Cigler, P., More, G., Bize, P., Meier, C. M., Frey, C. F., Basso, W. & Keller, S., 2024, Trypanosomiasis: An emerging disease in Alpine swift (Tachymarptis melba) nestlings in Switzerland?, International Journal for Parasitology: Parasites and Wildlife 23, pp. 100895-100895 : 100895-

2.4.1. Trypanosoma sp. morphometrics

Trypomastigotes (between 6 and 15 per sample) found in three samples from 2022 were measured. The smears were evaluated using a Nikon Eclipse Ci microscope and photographed with a calibrated Nikon Camera (model DFK 23 UP 031). Measurements were taken with the NIS- Elements D software (Version 5.02.03 64 bit) and included: total length without flagellum (PA), posterior end to centre of kinetoplast ( PK), posterior end to centre of nucleus ( PN), centre of nucleus to anterior end (NA), centre of kinetoplast to centre of nucleus ( KN), body width at the centre of the nucleus ( BW), length of free flagellum ( FF), area of the trypomastigote ( AT) and area of the nucleus ( AN) ( Bennett et al., 1994).

3. Results

3.1. Field observations

The affected nestlings showed pale coloration of mucous membranes and feet, alongside moderate to severe bruising across the body. These signs were observed earliest in nestlings aged 14–20 days, although most observations were in nestlings aged 20–45 days. Other changes in the form of primary feather abnormalities, including stunted growth, retained feather shafts and/or blood keels, were observed in nestlings aged 40 days and over ( Fig. 3 ). However, as these changes were not noted consistently, the exact numbers are unknown.

A considerable louse fly burden was described on both healthy and affected nestlings, with a mean of 5.5 ± 0.2 per nestling throughout development (690 observations). The highest burden on an individual nestling observed in 2022 was 39 louse flies.

The numbers of fledged, deceased or missing (but presumed dead) nestlings, as well as the average age and weight at the time of death were evaluated for colonies A, B and C and compiled in Table 1.

3.2. Post-mortem examination and histopathology

All necropsied nestlings (n = 5) showed identical findings on gross pathology, including extensive subcutaneous haemorrhages and pale discoloration of mucous membranes and feet. The haemorrhages were randomly distributed; however, were more prominent along the back, the top of the head, both sides of the wings and on the breast ( Fig. 4 ). In animals sampled within 24 h of death, myocardial petechiae could be observed (n = 2). Significant splenomegaly was present in all animals. All birds were in good body condition, had considerable fat reserves and full intestinal tracts. One bird had significantly shorter primary feathers on one wing, without indication of a trauma. The remainder of the plumage and the uropygial gland were unremarkable.

Histopathology (n = 3, one from each colony) confirmed extensive areas of haemorrhage in subcutaneous tissue, reaching into the underlying musculature. The epidermis and feather follicles were without abnormality. Pectoral and wing (mainly biceps) musculature showed multifocal, interstitial, often perivascular accentuated small to medium sized infiltrations of mononuclear inflammatory cells (macrophages, lymphocytes and plasma cells, Fig. 5A /B). In two birds, multifocal haemorrhaging was observed along the epicardium and, in part, infiltrating into the underlying myocardium. Sporadically distributed throughout the myocardium, a mixed inflammation composed of macrophages, lymphocytes, plasma cells and few heterophils, was seen in all three birds.

In proximity to the inflammatory foci, 4–5 μm, round-to-oval structures containing two basophilic nuclear-like structures were observed. These structures resembled the amastigote stages of kinetoplastid parasites and were found primarily in skeletal and cardiac musculature, as well as sporadically dispersed amongst fat cells in connective tissue and appeared to be extracellular ( Fig. 5C ). No trypomastigotes could be identified within blood vessels.

The bursa fabricii examined in two birds demonstrated a depletion of the medullary follicles characterized by moderate to severe lymphocytolysis, surrounded by a distinct epithelium ( Fig. 5D ). The spleens were difficult to assess due to autolytic changes. The lymphoid follicles were hypocellular and could not be clearly discriminated from the perivascular sheets, indicative of a mild lymphoid depletion.

All other organs were generally unremarkable with no indications of any other underlying diseases.

3.3. Ancillary diagnostics

All birds (n =5) tested negative for both West Nile and Usutu viruses. Both birds examined for Polyoma- and Circovirus tested negative.

A bacteriological examination of liver, lung, and spleen in one nestling resulted in growth of unspecific flora interpreted as post-mortem overgrowth.

3.4. Molecular diagnostics

The initial Leishmania spp. PCR ITS1 resulted positive in all three samples with an approximate 350 bp length band, and thus larger than expected for Leishmania spp. Sequencing analysis from one of these samples (consensus sequence of 260 bp) showed 97–100% identity with several Trypanosoma spp. 18S rRNA sequences, but with a low coverage (14–16%).

Tissue pools of the nestlings (3/3) were positive for Trypanosoma spp. based upon results from the 18SrRNA PCR (gel not shown).The amplicon from one nestling was sequenced and the consensus sequence of 539 bp (primers trimmed GenBank accession no. OR598759) which showed an identity of 99.63% with 100% coverage with several sequences: Trypanosoma corvi (JN006854 and AY461665, from Buteo from Czechia and Corvus frugilegus from the United Kingdom, respectively), Trypanosoma sp. (LZ 2011, JN006841, from Ficedula albicollis in Czechia) and Trypanosoma sp. AAT (AJ620557, from Strepera sp. , an Australian bird). Our sequence showed two mismatches with all the referenced sequences.

3.5. Evaluation of blood smears

Of the 72 evaluated blood smears from the 2022 season (colony B and C), 65 were from nestlings which fledged at the end of the season (90.2%). Overall, 27.8% of samples contained trypomastigotes (20/72). Seven animals died after 45 days-of-age, of which six (85.7%) had trypomastigotes and one did not (14.3%). Of the nestlings that fledged (n = 65), 14 had trypomastigotes (21.5%) visible on blood smear.

Forty blood smears were examined in detail. An average trypomastigote burden of 7.9 trypomastigotes per 10 HPF was observed amongst positive animals (n = 20). In the sample with the highest burden, 56.4 trypomastigotes per 10 HPF were found. In most smears, the trypomastigotes were observed in clusters of rosette-like formations ( Fig. 6 ).

No clear difference was observed in the thrombocyte counts between positive and negative nestlings, with an average of 13.9 thrombocytes per 10 HPF.

Positive nestlings showed a higher level of mononuclear cells (18.6 per 10 HPF) in comparison with negative ones (8.2 per 10 HPF). Positive nestlings also showed a slightly higher level of granulocytes (3.9 per 10 HPF) when compared to negative ones (2.1 per 10 HPF). All counts, including standard deviation (SD), are compiled in Table 2 and Fig. 7.

3.6. Trypomastigote morphometrics

Trypomastigote morphometrics were similar in all evaluated blood smears ( Fig. 6C ). The total length without the flagellum was on average 30.0 μm, based on n =36 from three blood smears, compiled in Table 3 .

Figures

Fig. 3. Missing (left wing) and poor quality (right wing) primary feathers on a 45-day-old nestling.

Fig. 4. Distribution of louse flies on an Alpine swift nestling (A) compared with the distribution of bruising on post-mortem examination with plumage removed (B).

Fig. 5. Skeletal musculature of a nestling Alpine swift showing infiltrations of mononuclear inflammatory cells (A, B) and presumably extracellular, amastigote-like structures (C). Bursa fabricii of a nestling Alpine swift with depletion of the medullary follicle with lymphocytolysis (asterisk) and a distinct epithelium (arrows) (D).

Fig. 6. Blood smears of nestling Alpine swifts with high (A) and moderate (B) trypomastigote burdens. Close-up of a trypomastigote between erythrocytes (C).

Fig. 7. Box plots comparing average counts per 10 HPF of granulocytes, mononuclear cells, and thrombocytes between positive (n =20) and negative (n =20) 45- day-old Alpine swift nestlings sampled in 2022.