Retrospective study of canine infectious haemolytic anaemia cases reveals the importance of molecular investigation in accurate postmortal diagnostic protocols.

Infectious haemolytic anaemia (IHA) in dogs share similar clinical signs including fever, lethargy, icterus, paleness of mucous membranes and splenomegaly. Postmortal findings are similar and, without additional diagnostic methods, an accurate aetiological diagnosis is difficult to achieve. In order to investigate causes of lethal IHA in Croatian dogs, we performed a retrospective study on archived formalin-fixed, paraffin-embedded tissue blocks (FFPEB) from dogs that died due to haemolytic crisis, using microscopic and molecular diagnostic tools to determine the aetiological cause of disease. Molecular analysis was performed on kidney, lung, myocardium and spleen on FFPEB from all dogs. The originally stated aetiological diagnosis of B. canis or leptospirosis was confirmed in only 53% of the dogs. PCR and sequencing revealed that, in addition to the expected pathogens, B. canis and Leptospira interrogans, the presence of previously undiagnosed "new" pathogens causing anaemia including Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum. Furthermore, Theileria capreoli was detected for the first time in a dog with postmortal descriptions of lesions. Intensive extravascular hemolysis was noticeable as jaundice of the mucosa, subcutis and fat tissue, green or yellow discoloration of renal parenchyma caused by bilirubin excretion in the renal tubules and bile accumulation within the liver in 90% of the dogs. This work highlights the value of molecular diagnostics to complement traditional ante-mortem and post-mortem diagnostic protocols for the aetiological diagnosis of pathogens associated with IHA.

Microscopic and molecular analysis of Babesia canis in archived and diagnostic specimens reveal the impact of anti-parasitic treatment and postmortem changes on pathogen detection.

BACKGROUND: Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. METHODS: The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. RESULTS: The morphology and size of merozoites from live dogs corresponded to that of previously described 'large' Babesia. The morphology and size of merozoites were significantly different (P < 0.001) in postmortem samples, however, and more consistent in shape and size with piroplasm cells previously referred to as 'small' Babesia. PCR and sequencing confirmed B. canis as the causative agent of disease in all investigated dogs, including in postmortem negative tissue imprints from dogs treated at least 24 h before death. CONCLUSIONS: Changes in the morphology of 'large' B. canis to 'small'-like Babesia observed by light microscopy appear to represent a common postmortem change. Classification of Babesia parasites into 'large' and 'small' Babesia using only microscopy of postmortem slides should be treated with caution. PCR-based methodologies for detection and molecular typing of Babesia spp. may prove valuable for investigating suspected cases of babesiosis following necropsy.