Piroplasmida, Wenyon

Yam, Jerald, Gestier, Sarah, Bryant, Benn, Campbell-Ward, Michelle, Bogema, Daniel & Jenkins, Cheryl, 2018, The identification of Theileria bicornis in captive rhinoceros in Australia, International Journal for Parasitology: Parasites and Wildlife 7 (1), pp. 85-89 : 86-88

2.3. Piroplasmida PCRs

DNA extractions were conducted using the DNeasy blood and tissue DNA extraction kit according to manufacturer's protocol (Qiagen). T. orientalis qPCR was conducted according to a previously described multiplex hydrolysis probe qPCR assay (Bogema et al., 2015). A generic PCR assay targeting the 18S rRNA gene of the Piroplasmida was also conducted using previously described primers Piroplasmid-F: 5′- CCAG CAGCCGCGGTAATT-3′ and Piroplasmid-R: 5′-CTTTCGCAGTAGTTYG- TCTTTAACAAATCT-3’ ( Tabar et al., 2008; Baneth et al., 2013). Amplification was carried out with the BIOTAQ™ DNA Polymerase kit (Bioline) to make up a 25 μL PCR cocktail containing: 1 X BIOTAQ buffer, 1.5 mM MgCl 2, 0.2 mM dNTPs, 0.4 μM of each primer, 1 unit of BIOTAQ DNA polymerase and molecular grade water. PCR was carried out with the following thermal cycling parameters: Initial denaturation 94 ̊C for 3 min followed by 35 cycles at 94 ̊C for 30 s, 64 ̊C for 45 s, 72 ̊C for 30 s with a final extension of 72 ̊C for 7 min. Full length Theileria bicornis 18S rRNA gene was amplified with primers 18 SAN, 18SBN ( Nijhof et al., 2003) and another pair of internal primers designed for this study, 18S-F1: 5′-GATCCTGCCAGTAGTCATATG-3′ and 18S-R1: 5′-TACTCCCCCCAGAACCCA-3’. PCR products were viewed on a 1.5% agarose 0.5 × TBE gel stained with GelRed (Biotium, USA), purified with QIAquick PCR purification kit (Qiagen) and subjected to Sanger sequencing with the primers described above.

2.4. Molecular phylogeny

T. bicornis 18S rRNA sequences were aligned using Geneious version (7.1.9) (Kearse et al., 2012) and subjected to a nucleotide BLAST comparison with the Genbank database. Phylogenetic analysis was conducted using DNAdist within the PHYLIP package (Felsenstein, 2005) and a neighbour-joining tree was generated with 1000 bootstrap replicates to estimate phylogenies ( Fig. 2 ). The analysis included 19 Theileria spp. , six Babesia spp. , one Cytauxzoon sp. and Toxoplasma gondii as the outgroup.

3. Results

3.1. Blood smear examinations

Piroplasms were observed on blood smears from Aluka, but in Umfana, there were observations of red cell inclusions reflected by dot forms, signet ring forms and also rod forms on Giemsa-stained smears, but not Diff- Quik-stained smears. Given that Umfana was an asymptomatic animal, the clinical significance of these inclusions was doubtful.

Serum biochemistry results from the D. bicornis cohort were generally unremarkable, with minor elevations in alkaline phosphatase and aspartate aminotransferase (AST) in some animals. Siabuwa additionally displayed elevated creatine kinase (CK; 1154 U/L, reference range 142–742 U/L) and blood urea nitrogen (BUN; 9.0 mmol/L, reference range 2.5–8.1 mmol/L). Siabuwa was positive for piroplasms on blood smear ( Fig. 1 ), while all other D. bicornis tested negative. Piroplasm morphology resembled and was in the size range (1.5 μm) of Theileria spp (Izzo et al., 2010). rather than Babesia spp. and was generally of the comma-shaped form ( Fig. 1 ), although ring forms were also occasionally observed.

3.2. PCR amplification and sequencing

All animals tested negative for Theileria orientalis . Despite a negative blood smear, Aluka tested positive for Piroplasmida 18S rRNA using a generic piroplasmid PCR. Umfana, the asymptomatic male C. simum which returned a positive blood smear, also tested positive for piroplasmids. Of the seven D. bicornis tested, only one (Siabuwa) returned a positive PCR result for Piroplasmida , which was also consistent with the blood smear results. Sequencing of the 18S rRNA amplicons revealed the presence of T. bicornis in all three samples. Additional PCR amplification of the 18S rRNA gene resulted in 1729 bp, 1691 bp and 1616 bp of sequence from the samples of white rhinoceros Umfana (MF536661), Aluka (MF536660) and black rhinoceros Siabuwa (MF536659) respectively.

3.3. Molecular phylogeny

Phylogenetic analysis of 1474 bp of the 18S rRNA gene of the C. simum and D. bicornis piroplasmid strains demonstrates the close relationship to T. bicornis ( Fig. 2 ). Furthermore, the T. bicornis cluster falls outside the non-transforming and transforming Theileria groups along with Cytauxzoon felis and Theileria equi ( Fig. 2 ), which is consistent with prior studies ( Nijhof et al., 2003; Schreeg et al., 2016). Alignment of the 18S rRNA genes also revealed the presence of two haplotypes of T. bicornis H2, which was previously described ( Otiende et al., 2016) and alignment of the 396 bp T. bicornis haplotypes revealed a new T. bicornis haplotype, H4, identified in this study (see Figs. 2 and 3 ). The T. bicornis haplotype in the two infected white rhinoceros, Aluka and Umfana was 100% homologous to haplotype H2; however, a new haplotype, H4, was identified in D. bicornis . H4 (accession number MF567493) in the infected black rhinoceros (Siabuwa) is 99% homologous to the H2 (accession number KC771141).

4. Discussion

In this study, we examined blood samples from captive C. simum and D. bicornis housed at Taronga Western Plains Zoo, Australia for piroplasmid parasites. Haemoparasites such as trypanosomes ( McCulloch and Achard, 1969; Mihok et al., 1992a,b) and Babesia bicornis ( Nijhof et al., 2003) have been associated with rhinoceros mortalities in Africa and were considered in the differential diagnosis of a 16 yr old female C. simum ; however, neither of these parasites was detected. Theileria bicornis was detected in blood samples from both dead (Aluka) and asymptomatic (Umfana) C. simum via PCR; however, piroplasms were only observed in blood films from Umfana, suggesting that the T. bicornis infection in Aluka was of a low intensity. The pathogenic potential of T. bicornis is not fully understood as this organism has been poorly studied; however asymptomatic infections are common in both black and white rhinoceroses in Africa ( Otiende et al., 2015). Only a single case of T. bicornis infection has been recorded in association with rhinoceros deaths although it presented as a coinfection with B. bicornis ( Nijhof et al., 2003; Otiende et al., 2015). Thus, the presence of T. bicornis was considered an incidental finding unlikely to be involved in the C. simum mortality.

PCR screening of the captive population of D. bicornis for piroplasmids revealed only a single T. bicornis -positive animal (Siabuwa), which was also confirmed by blood smear. This animal had elevated AST and CK levels that were potentially linked to tissue damage, but blood parameters were otherwise normal. The blood profile of Siabuwa was similar to a piroplasm prevalence study in white rhinoceroses by Govender et al. (2011) where blood parameters of the animals had no significant changes as well. Siabuwa was translocated from Africa as were the two T. bicornis -positive C. simum , while the remaining 6 captive-bred D. bicornis were T. bicornis negative. This suggests that T. bicornis was introduced to Australia with wild-caught rhinoceros and that transmission amongst the Australian captive population did not occur. Currently, the vectors identified to be capable of transmitting T. bicornis are Dermacentor rhinocerinus and Amblyomma rhinocerotis (Knapp et al., 1997; Otiende et al., 2016). These ticks are present in Africa, have not been identified in Australia and are characterized by long, sturdy mouthparts capable of penetrating the thick rhinoceros hide (Horak et al., 2017). Whether endemic Australian tick species are competent vectors of T. bicornis and other rhinoceros blood parasites is unclear, but they may lack the necessary mouthparts to achieve transmission. Transplacental transmission has been demonstrated for several piroplasmid species ( Phipps and Otter, 2004; Fukumoto et al., 2005; Mierzejewska et al., 2014; Zakian et al., 2014; Sudan et al., 2015; Swilks et al., 2017) but tends to occur only at low frequencies and there was no evidence from this study that T. bicornis was transmitted via this route within the captive population.

Sequence alignments of H2 and H4 revealed a difference of a single nucleotide substitution and a single thymine nucleotide insertion. Current literature of T. bicornis haplotype indicates H1 and H3 to only occur in black rhinoceros and H 2 in white rhinoceros ( Table 1 in Otiende et al., 2016). Whether haplotype H4 only occurs in black rhinoceros or any of these haplotypes contributes to disease remains unknown, but further studies could be done to determine haplotype specificity for a particular host or if the haplotypes play a pathogenic role in infected animals. Phylogenetic analysis placed the T. bicornis clade in this study outside the transforming and non-transforming clades of the Theileria group ( Fig. 2 ). T. bicornis appears to be close relatives of C. felis , T. youngi , B. bicornis and T. equi which are consistent to previous studies ( Nijhof et al., 2003; Otiende et al., 2016). The complete lifecycle of T. bicornis has not been established but it has similar characteristics to the non-transforming Theileria group suggesting it may be a largely benign parasite ( Nijhof et al., 2003). However as both horses and rhinoceros are odd-toed ungulates classified under the order Perissodactyla , it is worth to noting that T. equi that causes clinical equine piroplasmosis ( Nijhof et al., 2003; Laus et al., 2015; Otiende et al., 2015, 2016), was recently detected in rhinoceros (Govender et al., 2011).

Piroplasmids have coevolved with their hosts ( Otiende et al., 2015). Benign infections are common in non-transforming Theileria species, for example T. orientalis genotype Buffeli in cattle (Kamau et al., 2011) and T. velifera ( Uilenberg, 1981; Mans et al., 2015). Some of these haemoparasites can persist as lifelong infections in the host, only causing clinical signs when the animals are immunosuppressed or undergo stress from translocation, rearing conditions or pregnancy ( Sugimoto and Fujisaki, 2002). Translocation of the rhinoceros in Africa to suitable and safe environments is integral for the conservation of these magnificent animals. However, translocation stress has been reported to decrease PCV levels (Kock et al., 1999) and is also linked to immune suppression in the animals which can lead to morbidity and/or fatality (Glaser and Kiecolt-Glaser, 2005; Martin, 2009; Otiende et al., 2015). Thus screening of animals for haemoparasites prior to translocation would be prudent for future breeding programs.

5. Conclusion

We revealed for the first time in Australia the presence of T. bicornis in both white and black rhinoceros. Evidence from this study suggests that the parasite was acquired in Africa and was not transmitted within the captive rhinoceros population within Australia. A new T. bicornis haplotype, H4, has been identified. T. bicornis infection intensity was low and haematological parameters within infected rhinoceros were unremarkable suggesting that infection with this parasite was likely incidental rather than the cause of the 2012 white rhinoceros mortality event. However, given that translocation-induced stress is a major trigger factor for theileriosis; future screening of translocated rhinoceros would be prudent to ensure successful breeding programs.

Figures

Fig. 2. Molecular phylogenetic analysis of the piroplasm 18S rRNA gene, including the three rhinoceros samples (Aluka, Umfana and Siabuwa) used in this study. The 18S rRNA sequences were extracted from Genbank during BLAST analysis. The rooted phylogenetic tree was constructed using the neighbour-joining method with T. gondii forming the outgroup. Bootstrap percentages are represented on each node based on 1000 replicates. Phylogenetic analyses were conducted using the PHYLIP packages (Felsenstein, 2005). The haplotypes of the three T. bicornis sequences from this study are indicated at the end of the sequence label.

Fig. 1. Photomicrograph of Diff-Quik stained blood smear from black rhinoceros, Siabuwa infected with T. bicornis. The morphology of T. bicornis is not well-described in the literature. We observed comma-shaped piroplasms approximately 1.5 μm in length closely resembling T. orientalis. Ring forms were also occasionally observed (not shown). Bar = 5 μm.

Fig. 3. Truncated alignment of the 18S rRNA T. bicornis haplotypes including the new haplotype H4 identified in this study. Geneious version (7.1.9) (Kearse et al., 2012) was used to generate alignments to highlight the differences between the four T. bicornis haplotypes. 18S rRNA sequences of the three previously described T. bicornis haplotypes H1 to H3 (accession numbers KC771140 to KC771142 respectively) were extracted from Genbank for this analysis. The new T. bicornis haplotype H4 was submitted to Genbank and assigned accession number MF567493.