A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis)

Abstract

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100% sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

Keywords: Anaemia; Babesia; Kangaroo; Piroplasm; Wallaby.

Graphical abstract

Fig. 1

Map showing the distribution of the 38 cases of Babesia infection in eastern grey kangaroos in coastal New South Wales and southeastern Queensland over the period 1995–2013. Insert also shows the two locations of the three cases identified in agile wallabies in northern Queensland in 2009 and 2013. The locations of cases were converted to GPS coordinates and mapped using GPS Visualizer on 21/05/2014 (www.gpsvisualizer.com).

Fig. 2

Photomicrographs showing the forms of Babesia seen in cytological preparations and tissue sections in macropods. (A) Agile wallaby. Giemsa stained peripheral blood smear showing extraerythrocytic zoites (thin arrow) and merozoites (thick arrow) within an intact erythrocyte. (B) Eastern grey kangaroo. Diff-Quik-stained renal impression smear demonstrating 2 or 4 merozoites within intact erythrocytes (thick arrows) and clusters of extraerythrocytic zoites (thin arrows). (C) Eastern grey kangaroo. Diff-Quik-stained brain squash preparation showing large clusters of intravascular zoites (arrows). (D) Eastern grey kangaroo. H&E stained section of kidney glomerulus showing merozoites within intact erythrocytes (thick arrows) and as large extraerythrocytic clusters of zoites (thin arrows). All scale bars = 20 µm.

Fig. 3

Transmission electron micrographs showing the intravascular location and structure of Babesia organisms in the kidney and brain of eastern grey kangaroos. (A) Kidney, the cytoplasm of two adjacent erythrocytes contains Babesia merozoites (arrows) with a membrane-bound nucleus (N) and cytoplasm containing polymorphic vacuoles and some electron dense particles (C) (scale bar = 1.0 µm). (B) Brain, adjacent to an intact erythrocyte and the nucleus of an endothelial cell is a cluster of extraerythrocytic Babesia organisms containing electron dense micronemes and developing pellicles (arrows) (scale bar = 2.0 µm). (C) Brain, within the capillary lumen is a cluster of eight or nine extraerythrocytic organisms (thick arrow) and a distorted erythrocyte (thin arrow) containing four intracytoplasmic parasites (scale bar = 5.0 µm).

Fig. 4

Phylogenetic tree of 18S ribosomal RNA (18S rRNA) gene sequences of eastern grey kangaroo and agile wallaby Babesia and other piroplasms that are in the GenBank nucleotide database. For each sequence, the GenBank GI number is followed by the species name. The representative Babesia isolates from eastern grey kangaroos and an agile wallaby in this study are shown with a ▪ and a ♦ respectively. Evolutionary history was inferred using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood (−1820.5242) is shown. Initial tree for the heuristic search was obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise, BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The scale bar represents the number of substitutions per nucleotide. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2011).

Fig. 5

Phylogenetic tree of heat shock protein 70 (hsp70) gene sequences of eastern grey kangaroo and agile wallaby Babesia and other piroplasm hsp70 sequences in the GenBank nucleotide database. For each sequence, the GenBank GI number is followed by the species name. The representative Babesia isolates from eastern grey kangaroos and an agile wallaby in this study are shown with a ▪ and a ♦ respectively. The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (−6290.3774) is shown. Initial tree for the heuristic search was obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise, BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The scale bar represents the number of substitutions per nucleotide. All positions containing gaps and missing data were eliminated. There are limited data available on this locus within the public data repositories and as such there is some lack of consistency with the 18S ribosomal RNA tree.