Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization

Abstract

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

Emergence and Epidemiology of Bovine Babesiosis Due to Babesia divergens on a Northern German Beef Production Farm

“…All four animals were in good body condition, showed multifocal subcutaneous oedemas, mild jaundice, splenomegaly, haemoglobinuric nephrosis and dark brown urine. Infestation with adult and nymphal I. ricinus was also noted (19).…”

“…In the blood smears of the two symptomatic animals treated in the Clinic for Cattle, typical small Babesia divergens merozoites, located at the periphery of erythrocytes, were detected (Figure 2). Parasitaemia amounted to 37.0 and 39.3%, respectively (19). Blood samples were also subjected to PCR and Babesia divergens infection was confirmed, as obtained partial 18S rRNA sequences showed 100% nucleotide identity to published B. divergens sequences (e.g., LC477143, 100% query cover).…”

“…The beginning of the outbreak has been briefly summarized previously in an overview on babesiosis diagnosis and management for local veterinarians (18) and the development of an in situ hybridization for post mortem diagnosis (19). In detail, haemoglobinuria was first noted in one of the farm's herds, consisting of 56 animals, on June 3rd 2018.…”

“…On clinical examination, an elevated body temperature and increased heart rate (140/min, pounding beat) with a physiological respiratory rate (36 breaths/min) were noted. The rumen was hypo-motile and visible mucous membranes were pale and jaundiced (19).…”