The first confirmed cases of pigeon rotavirus A (RVA) infection in domestic pigeons (Columba livia) in Poland

From 2011 to 2020, archived liver samples from 117 pigeons (66 fancy and 51 racing birds) originating from 74 individual lofts were tested for the presence of pigeon-type RVA. Six of these samples were formalin-fixed paraffin-embedded (FFPE) material, and the remaining samples had been frozen when fresh. They originated mainly from the Mazovia voivodeship (n = 89), but also from Warmia and Mazury (n = 9), Pomerania (n = 4), Łódź (n = 2), Silesia (n = 1) and Greater Poland voivodeships (n = 1). The region of origin was unknown for eleven individuals (Supplementary Table 1). A more detailed necropsy was performed on four pigeons from three lofts and a histopathological examination was carried out on three pigeons from these lofts. Clinical data was also provided by the breeders of these four pigeons. They were approximately four-month-old racing pigeons that were delivered for necropsy in July 2017 and August 2018. They originated from three different lofts from the Łódź, Silesia and Mazovia voivodeships.

Necropsy and sample collection were performed for all pigeons according to standard avian necropsy protocols in the Department of Pathology and Veterinary Diagnostics in the Institute of Veterinary Medicine of the University of Life Sciences in Warsaw. Livers were collected in sterile tubes and stored at -20°C until extraction of nucleic acids was carried out. For histopathological examination, samples of the heart, liver, kidney, spleen, duodenum and pancreas were collected during necropsy from three pigeons submitted in 2017 and 2018 from three different outbreaks. They were fixed in 10% buffered formalin and embedded in paraffin following standard protocols. The paraffin blocks were cut into 5 μm sections, stained with haematoxylin and eosin and examined using light microscopy.

Total RNA was extracted from comparable pea-sized sections of frozen liver samples using a NucleoMag VET kit (Macherey-Nagel, Düren, Germany) with a KingFisher Flex Purification System (Thermo Fisher Scientific, Waltham, MA, USA). Ribonucleic acid from FFPE samples was extracted using a miRNeasy FFPE Kit (Qiagen, Hilden, Germany). All procedures were performed according to the manufacturers’ instructions. A defined number of copies of in vitro-transcribed RNA of the egfp enhanced green fluorescent protein gene was added to each sample during RNA extraction as an internal control to confirm the efficiency of RNA extraction and the RT-qPCR reaction (11). Screening for pigeon RVA was performed with a previously published VP6-specific RT-qPCR assay (forward primer: CoRVA_VP6_868+, 5´-GCCCGYAATTTCGATTCAATACG-3´; reverse primer: CoRVA_VP6_943-, 5´-GTGCTGCYACTCCAGGTGTCAT-3´; TaqMan probe: CoRVA_VP6_898_P, 6FAM-5´-TTCCAACTTGTTAGGCCRCCAA-3´-BHQ1) (22) with AgPath-ID One-Step RT-PCR reagents (Thermo Fisher Scientific). Briefly, 5 μL of extracted RNA was mixed with 2 μL of primer mix (containing each primer at a concentration of 5 μM) and denatured at 95°C for 5 min. Subsequently, 18 μL of master mix containing 12.5 μL of AgPath 2× buffer, 2 μL of AgPath enzyme mix (reverse transcriptase and Taq polymerase), the RVA-specific probe at a final concentration of 0.12 μM and primers and probes for the detection of egfp RNA was added and the reaction was performed with the following cycler setup: 45°C for 10 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. Standard preparations of cell culture supernatant and extracted RNA from isolates DR-7 and DR-5 (22), respectively, served as respective positive controls of RNA extraction and RT-qPCR. Cycle of quantification values were calibrated by adjusting the threshold to set the Cq values of the two positive controls to constant values (+/− 0.5) in each RT-qPCR run. Values lower than 30 were considered as indicative of an acute RVA infection (22, 23).

Partial VP6 gene sequences were generated for all RVA-positive pigeons with a Cq value <30. A 438-base pair (bp) fragment of the VP6 segment was amplified by conventional RT-PCR using AvRVA_VP6_243+ (5′-GGAYGCGAATTACGTTGAGAATG-3′) and CoRVA_ VP6_680- (5′-TTTAAYACTCTTCGCATTGGTAC-3′) primers (22). Following gel electrophoresis, products of the expected length were extracted using Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). Sanger sequencing was performed by Eurofins Genomics (Ebersberg, Germany). Raw sequences were trimmed for quality and primer sequences to achieve a final sequence of 392 bp. Sequences were submitted to GenBank under accession numbers OR400329 to OR400352.

Phylogenetic analysis was performed for all partial VP6 sequences generated during this study together with avian RVA VP6 sequences derived from GenBank. Following nucleotide sequence alignment using MUSCLE, a phylogenetic tree was built using the neighbour-joining algorithm and Jukes–Cantor distance model in Geneious Prime 2021.0.1 (Biomatters Ltd., Auckland, New Zealand). Avian RVA VP6 sequences of genotypes I11 and I21 were used to root the tree.

Using a VP6-specific RT-qPCR test, different levels of pigeon-type RVA RNA could be detected in 57 out of 117 samples (48.7%) (Fig. 1A, Supplementary Table 1). Based on previous experiments, only calibrated Cq values below 30 were considered indicative of an acute or recent RVA infection, whereas calibrated Cq values above 30 were considered as possibly indicating remnants of a past infection (22, 23). Such positive results were obtained for 24 out of 117 (20.5%) tested samples. Positive test results were obtained for 12 out of 51 (23.5%) samples from racing pigeons and 12 out of 66 (18.2%) samples from fancy pigeons (Fig. 1A). The positive pigeons originated from 19 out of the 74 tested lofts (25.7%). Positive samples were detected in the years 2014, 2015 and 2017 to 2019 (Fig. 1B, Supplementary Table 1). Outbreaks of RVA infection in racing pigeons were detected only between May and September, with most positive samples dating to August (Fig. 1C). The samples from fancy pigeons which were RVA positive were more evenly distributed over the year, with most positive samples submitted in December (Fig. 1C).

Fig. 1.

Detailed necropsy findings were available for four RVA-positive pigeons from three different outbreaks: birds A1 and A2 from the Łódź voivodeship, which were sick in 2018; bird B from the Silesia voivodeship, which was a case from 2018; and bird I from the Mazovia voivodeship, which was infected in 2017. Pigeons A1, A2, B and I had been tested positive for RVA by RT-qPCR. In two of these pigeons, the liver was moderately enlarged and congested (Fig. 2, photographs of pigeons A1 and B), and in the other two it was enlarged and mottled (Fig. 2, photographs of pigeons A2 and I). The kidneys of all four pigeons were enlarged; in one pigeon it was congested (Fig. 3, photograph of pigeon A1), and in two pigeons it was pale (Fig. 3, photographs of pigeons A2 and B). The spleen was pale and slightly or moderately enlarged in two pigeons (Fig. 4, photographs of pigeons B and I) and normal-sized and pale in one pigeon (Fig. 4, photograph of pigeon A1). The presence of food with water and bile was observed in the crop of three pigeons, and in these cases bile was also present in the proventriculus. The content of the intestines was green and mucoidal to watery in consistency. The content of the distal part of the gastrointestinal tract was dark green to brown and mucoidal. The vent and the surrounding feathers of two birds were stained with slimy faeces. In three pigeons, the air sacs showed varying degrees of opacity. Also in three pigeons, extensive areas of myocardial discoloration were found (Fig. 2, photographs of pigeons A2, B and I). The condition of the pigeons ranged from very good in two of the cases to satisfactory.

Fig. 2.

Fig. 3.

Fig. 4.

Microscopic analysis was performed for three RVA-positive pigeons (A1, B and I). Lesions observed in the liver were focal perivascular mononuclear infiltrates (Fig. 5 A1, Ba and Bb), necrosis (Fig. 5 Ba), congestion and haemorrhaging (A1), and steatosis and bile retention (not shown). In the kidneys, congestion, haemorrhaging, heterophilic perivascular infiltrates and accumulation in the epithelium of cells were seen (not shown). In one bird (I) bile in coronal fat tissue, myocardial congestion and focal necrosis were found (not shown).

Fig. 5.

Partial VP6 sequences were generated for all 24 RVA-positive pigeons. Phylogenetic analysis of partial VP6 sequences together with sequences from pigeons, domestic poultry and other species available in GenBank was performed and revealed all Polish sequences to belong to the pigeon-type RVA VP6 genotype I4 (Fig. 6). Six sequences detected in 2014 and 2015 clustered with sequences detected in Germany in 2010 to 2014 (subclade 2010, according to the nomenclature published by Rubbenstroth et al. (22)), whereas 14 Polish sequences from 2017 to 2019 clustered together with German sequences from the same years (subclade 2017b). Interestingly, a further sequence from 2015 and three sequences detected in 2018 formed two separate branches tentatively labelled subclades 2015b and 2018 (Fig. 6).

Fig. 6.