Material and methods
In early October 2019, mortality of free-living red-legged partridges was detected in a hunting estate in Cádiz (Southern Spain). Partridges in fair to poor body condition were found dead, or with severe central nervous signs, similar to those observed in the BAGV-related mortality in the same geographic area in 2010. The birds showed weakness, ruffled feathers, apparent blindness and neurological signs such as ataxia that led to loss of flight coordination or complete inability to fly.
We carried out full, detailed post-mortem examination on four individual birds that had died after showing neurological signs or were found dead and collected vascular growing feathers, heart, brain, liver, spleen and kidney for pathogen detection in addition to a complete set of tissues (heart, brain, spleen, liver, kidney, lung, skeletal muscle, skin, cecal tonsils, adrenal glands, gonads and pancreas) for histopathology and immunohistochemistry. No ethical approval was required as this study was based on samples from deceased animals.
Whole DNA and RNA were manually extracted from pulp of growing feathers, heart, brain, liver, spleen and kidney using TRI Reagent® (Merck Life Science, Madrid, Spain) according to manufacturer’s instructions. We tested for the presence of haemosporidian parasites using a nested PCR with posterior sequencing of the 524 bp PCR product (Waldenström et al., 2004). Sample RNA was analyzed using a generic flavivirus SYBR Green (BioRad, Madrid, Spain) real-time reverse transcription PCR as described previously (Moureau et al., 2007) and the new obtained sequences were deposited in GenBank (acc. n. OK424741, OK424742). We used ClustalW to align the sequences of the NS5 gene fragment obtained in this study (highlighted in bold in Figure 2) and reference sequences from GenBank including different variants of Bagaza virus, from different localizations (partial segment, 222 bp). A total of 30 sequences were used for this analysis, and sequence of West Nile virus was used as an outgroup. A phylogenetic tree was generated using MEGA version 10 (http://www.megasoftware.net) (Kumar et al., 2018) with the Maximum Likelihood method with Kimura 2-parameter distances (Kimura et al., 1980), this model was selected based on lowest values of Corrected Akaike Information Criterion (cAIC) and Bayesian Information Criterion (BIC). Reliability of internal branches was assessed using the bootstrapping method (1000 replicates).
In the case of avian malaria, phanerozoites observed in the hematoxilin-eosin-stained tissues were confirmed to be Plasmodiumspp. by immunohistochemistry (IHC) with the avidin-biotin-peroxidase complex (ABC) method (anti-Plasmodiumspp. polyclonal antibody; LSBio, Lifespan Biosciences, Seattle, Washington, USA). This method was also used to detect BAGV in different tissues, using a polyclonal anti-WNV antibody and following the protocol described by Gamino et al. (2013).