• View in gallery

    P. cynomolgi M strain and B. microti-like parasites in rhesus macaques. (A) P. cynomolgi parasitemias in rhesus monkeys Ri2011, Ri8012, Ri12103, and Ri12142 (solid lines) and B. microti-like parasitemia in Ri12142 (dotted line). Giemsa-stained thin film of Ri12142 showing (B) a P. cynomolgi trophozoite and (C) a B. microti-like “Maltese cross.”

  • View in gallery

    Phylogenetic tree constructed with (A) 18S rRNA and (B) β-tubulin sequences of the B. microti-like parasite detected in Ri12142 (MM-1) and various other Babesia parasite species. The number on each branch shows the percent occurrence in 1,000 bootstrap replicates.

  • 1

    Homer MJ, Aguilar-Delfin I, Telford SR 3rd, Krause PJ, Persing DH, 2000. Babesiosis. Clin Microbiol Rev 13 :451–469.

  • 2

    Bronsdon MA, Homer MJ, Magera JM, Harrison C, Andrews RG, Bielitzki JT, Emerson CL, Persing DH, Fritsche TR, 1999. Detection of enzootic babesiosis in baboons (Papio cynocephalus) and phylogenetic evidence supporting synonymy of the genera Entopolypoides and Babesia. J Clin Microbiol 37 :1548–1553.

    • Search Google Scholar
    • Export Citation
  • 3

    Emerson CL, Tsai CC, Holland CJ, Ralston P, Diluzio ME, 1990. Recrudescence of Entopolypoides macaci Mayer, 1933 (Babesiidae) infection secondary to stress in long-tailed macaques (Macaca fascicularis). Lab Anim Sci 40 :169–171.

    • Search Google Scholar
    • Export Citation
  • 4

    Gleason NN, Wolf RE, 1974. Entopolypoides macaci (Babesiidae) in Macaca mulatta. J Parasitol 60 :844–847.

  • 5

    Schuster FL, 2002. Cultivation of Babesia and Babesia-like blood parasites: agents of an emerging zoonotic disease. Clin Microbiol Rev 15 :365–373.

    • Search Google Scholar
    • Export Citation
  • 6

    Shayan P, Rahbari S, 2005. Simultaneous differentiation between Theileria spp. and Babesia spp. on stained blood smear using PCR. Parasitol Res 97 :281–286.

    • Search Google Scholar
    • Export Citation
  • 7

    Kawabuchi T, Tsuji M, Sado A, Matoba Y, Asakawa M, Ishihara C, 2005. Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan. J Vet Med Sci 67 :825–827.

    • Search Google Scholar
    • Export Citation
  • 8

    Tsuji M, Zamoto A, Kawabuchi T, Kataoka T, Nakajima R, Asakawa M, Ishihara C, 2006. Babesia microti-like parasites detected in Eurasian red squirrels (Sciurus vulgaris orientis) in Hokkaido, Japan. J Vet Med Sci 68 :643–646.

    • Search Google Scholar
    • Export Citation
  • 9

    Ruebush TK 2nd, Collins WE, Healy GR, Warren M, 1979. Experimental Babesia microti infections in non-splenectomized Macaca mulatta. J Parasitol 65 :144–146.

    • Search Google Scholar
    • Export Citation
  • 10

    Ruebush TK 2nd, Collins WE, Warren M, 1981. Experimental Babesia microti infections in Macaca mulatta: recurrent parasitemia before and after splenectomy. Am J Trop Med Hyg 30 :304–307.

    • Search Google Scholar
    • Export Citation
  • 11

    Chin W, Campbell CC, Collins WE, Roberts JM, 1983. Plasmodium inui and Babesia microti infections in the squirrel monkey, Saimiri sciureus. Am J Trop Med Hyg 32 :691–693.

    • Search Google Scholar
    • Export Citation
  • 12

    Cox FE, 1972. Protective heterologous immunity between Plasmodium atheruri and other Plasmodium spp. and Babesia spp. in mice. Parasitology 65 :379–387.

    • Search Google Scholar
    • Export Citation
  • 13

    Cox FE, 1978. Heterologous immunity between piroplasms and malaria parasites: the simultaneous elimination of Plasmodium vinckei and Babesia microti from the blood of doubly infected mice. Parasitology 76 :55–60.

    • Search Google Scholar
    • Export Citation
  • 14

    Matuschewski K, Mueller AK, 2007. Vaccines against malaria—an update. FEBS J 274 :4680–4687.

  • 15

    Wykes M, Good MF, 2007. A case for whole-parasite malaria vaccines. Int J Parasitol 37 :705–712.

 

 

 

 

Detection of New Babesia microti-like Parasites in a Rhesus Monkey (Macaca mulatta) with a Suppressed Plasmodium cynomolgi Infection

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  • 1 Biomedical Primate Research Centre, Department of Parasitology, Rijswijk, The Netherlands

A new type of piroplasm, phylogenetically closest to Babesia microti-like parasites previously detected in Eurasian red squirrels (Sciurus vulgaris orientis), was identified in a rhesus monkey (Macaca mulatta) imported from China. After challenge with Plasmodium cynomolgi M strain blood-stage parasites, the rhesus monkey repeatedly showed markedly reduced levels of Plasmodium parasitemia when compared with animals not infected with this organism.

Babesiosis is a tick-transmitted disease caused by intra-erythrocytic parasites of the genus Babesia. Babesial parasites are capable of infecting a wide variety of mammals, and awareness is growing of their role as zoonotic agent of human disease.1 Babesia-like parasites of the genus Entopolypoides macaci have been reported to infect nonhuman primates; based on phylogenetic analysis of small-subunit rRNA (SSUrRNA) sequences of this parasite and on serological and epidemiological data, it was suggested that the genus Entopolypoides is synonymous with that of Babesia.2 In primate centers, natural infections with this parasite have been shown in baboons (Papio cynocephalus),2 long-tailed macaques (Macaca fascicularis),3 and rhesus macaques (Macaca mulatta),4 providing animal models in species closely related to humans to study parasite–host relationships of this pathogen.

Here, we describe the detection and molecular characterization of a new Babesia-like parasite in a rhesus macaque and the effect of this infection on super-infection with the malaria parasite Plasmodium cynomolgi.

As the control arm of a study, 4 nonsplenectomized M. mulatta were inoculated intravenously with 1 × 106 P. cynomolgi M strain blood-stage parasites obtained from a donor monkey. Fingerpricks were taken every other day, and duplicate thin blood smears were made, one of which was Giemsa-stained to monitor the malaria infection. The research protocol was approved by an independent animal care and use committee and performed according to Dutch and European laws.

Unexpectedly, malaria peak parasitemia in one monkey, Ri12142, was significantly lower than in the other monkeys (Figure 1A), while other hematozoan parasites morphologically distinct from P. cynomolgi were observed in blood smears from this monkey (Figure 1, B and C). The parasites had a morphology resembling Babesia parasites, including, occasionally, the characteristic “Maltese cross” consisting of 4 pyriform-shaped trophozoites5 (Figure 1C). After P. cynomolgi peak parasitemia at Day 12 after infection, both parasitemias declined concurrently to low levels (Figure 1A). At Day 23, monkey Ri12142 was treated with chloroquine to cure the malaria. Two years later, Ri12142 was re-infected with P. cynomolgi. Again, morphologically distinct hematozoan parasites were observed, and the malaria infection was markedly suppressed compared with 3 other monkeys that had been infected from the same donor monkey (peak parasitemia 0.16% versus 3.13%, 2.48%, and 2.24% for the control monkeys; data not shown).

To molecularly characterize the piroplasm parasites in Ri12142, material was scraped with a clean scalpel from an unstained methanol-fixed blood smear obtained on Day 8 after both the first and second P. cynomolgi infections and transferred to a PCR tube for analysis; 75 μL of TE buffer was added, the material was heated for 15 min at 95°C, and, after a brief centrifugation, 5 μL was used as template in a PCR reaction with primers P7 and P8, specifically amplifying the hypervariable region V4 of the SSU rRNA genes of piroplasms.6 An amplified product was detected with a size of 439 bp in the material from both time points, more than 2 years apart. In contrast, PCR analysis of smears of the 6 monkeys that had received a P. cynomolgi blood-stage challenge at the same time as Ri12142 but that showed a historically “normal” parasite development did not show any product. Apparently, Ri12142 was the only monkey with a piroplasm infection, and this infection did not originate from either of the 2 donor monkeys.

For further molecular characterization, SSUrRNA was amplified using primers Piro0F and Piro6R for first round PCR and Piro1F and Piro5.5R for the nested PCR.7 The β-tubulin gene of the parasite was amplified using primers BmTubu93F and BmTubu897R.8 Products were cloned, and both strands of 5 independent clones for each gene were sequenced. The SSUrRNA and β-tubulin sequences have been deposited in GenBank (accession numbers EU168705 and EU168706, respectively).

Phylogenetic relationships were analyzed as described elsewhere,8 using sequences corresponding to regions 76–527 of AY693840 and 358–1085 of AB083377 of the SSUrRNA and β-tubulin genes, respectively. This revealed that the amplified piroplasm SSUrRNA product was closest (99.0% identity) to SSUrRNA of B. microti-like parasites that have been detected in Eurasian red squirrels (Figure 2A).8 Analysis of β-tubulin gave similar results (Figure 2B), with 97.3% identity to β-tubulin of B. microti-like parasites in Eurasian red squirrels.8 This parasite groups with the U.S.-type B. microti (= Babesia microti sensu stricto, regarded to be the major causative agent of human babesiosis), and therefore it was suggested that the squirrels may serve as an additional reservoir for the human babesiosis agent.8 This study shows that closely related B. microti-like parasites from this separate branch of B. microti can infect not only rodents but also non-human primates closely related to humans.

Previously, a partial SSUrRNA sequence (PB-1; Figure 2A) was published of a new B. microti-like species that infected baboons.2 The B. microti-like parasite SSUrRNA sequence from Ri12142 described here is clearly distinct from PB-1 (Figure 2A), revealing a new B. microti-like parasite that is capable of infecting a nonhuman primate.

The timing and origin of the B. microti-like infection are not known, and it is possible that the infection originates from the primate-breeding facility in Guangxi (China) from which the monkey had been imported 2 years before the first infection with P. cynomolgi. Long-term presence of B. microti is not uncommon, as previously reported in experimentally infected rhesus monkeys.9,10 The data presented here are in line with a report revealing that, in M. fascicularis, E. macaci parasitemias that had remained dormant for years were induced by stress,3 which in our case was likely induced by P. cynomolgi infection.

The B. microti-like infection of Ri12142 appeared to have had a marked suppressive effect on the P. cynomolgi parasitemia on 2 separate occasions. Data from a single monkey infected on 2 occasions did not constitute proof that this suppressive effect could be entirely attributed to the presence of the B. microti-like parasites. However, this is the first time that we have come across a suppressed P. cynomolgi infection in the more than 30 monkeys that we have infected to date. Reports on co-infections of Babesia and Plasmodium in non-human primates are scarce, and the effect of Babesia infection on Plasmodium parasitemia has not been elaborated.10,11 In one study, Entopolypoides in rhesus macaques did not suppress P. cynomolgi infection.4 These monkeys were, however, highly immunosuppressed due to splenectomy and antirhesus lymphocyte globulin therapy, most likely severely interfering with possible protective immune reactions. Interestingly, our data are in line with reports showing that mice that had recovered from infections with B. microti were protected against challenge with Plasmodium vinckei and Plasmodium chabaudi.12,13 The suppression of P. cynomolgi infection after chronic exposure to B. microti warrants further investigation of a possible protective role of Babesia infection on Plasmodium and underlying immune mechanisms, especially in light of renewed interest in the possibilities for development of attenuated vaccines for malaria.14,15

Figure 1.
Figure 1.

P. cynomolgi M strain and B. microti-like parasites in rhesus macaques. (A) P. cynomolgi parasitemias in rhesus monkeys Ri2011, Ri8012, Ri12103, and Ri12142 (solid lines) and B. microti-like parasitemia in Ri12142 (dotted line). Giemsa-stained thin film of Ri12142 showing (B) a P. cynomolgi trophozoite and (C) a B. microti-like “Maltese cross.”

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 4; 10.4269/ajtmh.2008.78.643

Figure 2.
Figure 2.

Phylogenetic tree constructed with (A) 18S rRNA and (B) β-tubulin sequences of the B. microti-like parasite detected in Ri12142 (MM-1) and various other Babesia parasite species. The number on each branch shows the percent occurrence in 1,000 bootstrap replicates.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 4; 10.4269/ajtmh.2008.78.643

*

Address correspondence to Alan W. Thomas, Biomedical Primate Research Centre, Department of Parasitology, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands. E-mail: thomas@bprc.nl

Authors’ addresses: Annemarie Voorberg-v.d. Wel, Clemens H. M. Kocken, Anne-Marie Zeeman, and Alan W. Thomas, Biomedical Primate Research Centre, Department of Parasitology, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands, E-mails: wel@bprc.nl,kocken@bprc.nl, zeeman@bprc.nl, and thomas@bprc.nl.

Acknowledgments: The authors thank members of the Animal Science Department of the BPRC for expert animal care and Ernst Verschoor for advice on phylogenetic analyses.

REFERENCES

  • 1

    Homer MJ, Aguilar-Delfin I, Telford SR 3rd, Krause PJ, Persing DH, 2000. Babesiosis. Clin Microbiol Rev 13 :451–469.

  • 2

    Bronsdon MA, Homer MJ, Magera JM, Harrison C, Andrews RG, Bielitzki JT, Emerson CL, Persing DH, Fritsche TR, 1999. Detection of enzootic babesiosis in baboons (Papio cynocephalus) and phylogenetic evidence supporting synonymy of the genera Entopolypoides and Babesia. J Clin Microbiol 37 :1548–1553.

    • Search Google Scholar
    • Export Citation
  • 3

    Emerson CL, Tsai CC, Holland CJ, Ralston P, Diluzio ME, 1990. Recrudescence of Entopolypoides macaci Mayer, 1933 (Babesiidae) infection secondary to stress in long-tailed macaques (Macaca fascicularis). Lab Anim Sci 40 :169–171.

    • Search Google Scholar
    • Export Citation
  • 4

    Gleason NN, Wolf RE, 1974. Entopolypoides macaci (Babesiidae) in Macaca mulatta. J Parasitol 60 :844–847.

  • 5

    Schuster FL, 2002. Cultivation of Babesia and Babesia-like blood parasites: agents of an emerging zoonotic disease. Clin Microbiol Rev 15 :365–373.

    • Search Google Scholar
    • Export Citation
  • 6

    Shayan P, Rahbari S, 2005. Simultaneous differentiation between Theileria spp. and Babesia spp. on stained blood smear using PCR. Parasitol Res 97 :281–286.

    • Search Google Scholar
    • Export Citation
  • 7

    Kawabuchi T, Tsuji M, Sado A, Matoba Y, Asakawa M, Ishihara C, 2005. Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan. J Vet Med Sci 67 :825–827.

    • Search Google Scholar
    • Export Citation
  • 8

    Tsuji M, Zamoto A, Kawabuchi T, Kataoka T, Nakajima R, Asakawa M, Ishihara C, 2006. Babesia microti-like parasites detected in Eurasian red squirrels (Sciurus vulgaris orientis) in Hokkaido, Japan. J Vet Med Sci 68 :643–646.

    • Search Google Scholar
    • Export Citation
  • 9

    Ruebush TK 2nd, Collins WE, Healy GR, Warren M, 1979. Experimental Babesia microti infections in non-splenectomized Macaca mulatta. J Parasitol 65 :144–146.

    • Search Google Scholar
    • Export Citation
  • 10

    Ruebush TK 2nd, Collins WE, Warren M, 1981. Experimental Babesia microti infections in Macaca mulatta: recurrent parasitemia before and after splenectomy. Am J Trop Med Hyg 30 :304–307.

    • Search Google Scholar
    • Export Citation
  • 11

    Chin W, Campbell CC, Collins WE, Roberts JM, 1983. Plasmodium inui and Babesia microti infections in the squirrel monkey, Saimiri sciureus. Am J Trop Med Hyg 32 :691–693.

    • Search Google Scholar
    • Export Citation
  • 12

    Cox FE, 1972. Protective heterologous immunity between Plasmodium atheruri and other Plasmodium spp. and Babesia spp. in mice. Parasitology 65 :379–387.

    • Search Google Scholar
    • Export Citation
  • 13

    Cox FE, 1978. Heterologous immunity between piroplasms and malaria parasites: the simultaneous elimination of Plasmodium vinckei and Babesia microti from the blood of doubly infected mice. Parasitology 76 :55–60.

    • Search Google Scholar
    • Export Citation
  • 14

    Matuschewski K, Mueller AK, 2007. Vaccines against malaria—an update. FEBS J 274 :4680–4687.

  • 15

    Wykes M, Good MF, 2007. A case for whole-parasite malaria vaccines. Int J Parasitol 37 :705–712.

Author Notes

Reprint requests: Alan W. Thomas, Biomedical Primate Research Centre, Department of Parasitology, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands, E-mail: thomas@bprc.nl.
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