Two cases of human babesiosis attributed to a B. divergens-
like agent in the south central region of the United States resulted in a fatal case in Missouri and a nonfatal but acute case in Kentucky (4
). Additionally, an acute case of babesiosis also attributed to a B. divergens-
like agent occurred in Washington State (10
). The organism in all three cases was described as morphologically and molecularly similar to B. divergens.
Further characterization of this parasite has been hampered by the small amount of original sample (4
) and no additional source of parasites. Recently, a Babesia
sp. of eastern cottontail rabbits was reported to have an SSU rRNA gene sequence identical to that of the Kentucky human agent (8
). The current study confirms this finding and, further, demonstrates that the Missouri isolate SSU rRNA gene sequence is also identical to that of the rabbit parasite. This study also shows the rabbit parasite to be morphologically indistinguishable from the Kentucky human parasite. Although eastern cottontails potentially provide a source of parasites, the circulating parasitemias are extremely low (8
); other experimental hosts have not been reported. The successful establishment of continuous in vitro cultures of this organism is not only the first culture isolation and establishment of a zoonotic Babesia
sp. from its natural host in the United States but also an important step toward further studies of this organism.
Continuous cultures of the cottontail rabbit Babesia
sp. were established in both cottontail rabbit and human erythrocytes with medium supplemented with human serum. Of the media tested for their abilities to support parasite growth, HL-1 medium best supported the parasite over extended culture periods, although MEM Alpha produced the highest parasitemias in cottontail erythrocyte cultures during early passages. In this study, neither domestic-rabbit serum nor FBS supplementation sustained parasite growth. Inasmuch as previous attempts in this laboratory to initiate primary cultures from infected cottontail rabbit blood in HL-1 medium supplemented with FBS were not successful (unpublished results) and a single attempt to initiate a culture in MEM Alpha supplemented with FBS in the current study failed, we concentrated our efforts toward finding a suitable serum alternative. Hence, although supplementation with heat-inactivated FBS for B. divergens
primary cultures has been reported (19
), this option was not explored in this study.
spp. are often cultivated in medium containing autologous serum and RBC of the vertebrate host, modeled after the culture system introduced by Levy and Ristic (18
). This strategy was not possible in the current study due to the lack of cottontail rabbit serum. Substitution of domestic New Zealand White rabbit (Oryctolagus cuniculus
) serum and RBC was attempted, but these did not support the parasite. In addition, media that successfully supported parasite growth in cottontail rabbit or human erythrocytes were unable to support the parasite in domestic-rabbit erythrocyte cultures. Although FBS has been shown to support in vitro growth of several Babesia
), as mentioned above, it did not support the cottontail rabbit parasite in this study.
Cottontail rabbit parasite cultures were established by using erythrocytes from two animals identified as positive for the presence of babesias by PCR, whereas our attempts to establish cultures from PCR negative blood were not successful (data not shown). From the primary cultures initiated using autologous erythrocytes, first passages into donor cottontail or human erythrocytes were generally successful when passaged from cultures in HL-1 medium supplemented with human serum. Early-passage parasites cultured in cottontail rabbit RBC in MEM Alpha did not readily adapt to subcultures with human RBC. However, by passage 5, the parasites were successfully introduced to human RBC, leading to the establishment of continuous cultures in human serum-supplemented medium with human erythrocytes. The morphologies of the parasites were similar whether the parasites were cultured in cottontail rabbit or human erythrocytes, as determined by light and transmission electron microscopy. This culture system provides a convenient in vitro protocol for maintaining a laboratory source of the cottontail rabbit parasites, since both human culture components are available from commercial sources.
The life cycles of the causative agents of acute babesiosis for patients in Missouri, Kentucky, and Washington are not known (4
). Given the molecular identity of the infecting parasite from the Kentucky case and that from Nantucket cottontail rabbits, it is likely that the vector tick is Ixodes dentatus
, which has been reported from much of the eastern United States; the closely related Ixodes spinipalpis
appears to replace I. dentatus
on lagomorphs in the western United States (16
). Accordingly, it is likely that babesiosis due to this parasite may occur in virtually any area of the United States. Our successful continuous laboratory propagation of this parasite will provide serological antigens, which will permit estimating its public health burden.