Isolation of Babesia divergens from carrier cattle blood using in vitro culture

Abstract

-Babesia divergens, the main causative agent of bovine babesiosis in Western Europe, was isolated from naturally infected cattle. Ninety-six blood samples were examined by means of an in vitro culture technique in sheep erythrocytes: 19 of them were collected from animals in the acute phase of the disease with visible parasitemia on blood smears, while the 77 remaining animals showed no microscopically detectable parasites. B. divergens was cultured from the 19 first blood samples as well as from 31 samples collected from asymptomatic animals. The time period before parasites could be detected in the culture varied in the latter samples from 6 to 20 days. The effects of sampling condition (anticoagulant used) and storage length were tested. A good correlation was obtained between immunofluorescent antibody test and culture, with identical results (positive or negative) for 89.6% of the samples collected from asymptomatic animals. The sensitivity of the in vitro culture method was determined and was about 10 parasites/mL of whole blood from three independent experiments performed with three different isolates, confirming its suitability to detect and culture diverse B. divergens isolates from carrier cattle. The parasites could indeed be isolated 9 months after the acute babesiosis phase in the blood of naturally infected animals. The 50 isolates collected in this study were successfully subcultured, cryopreserved and resuscitated using the same culture medium. The in vitro isolation of B. divergens from asymptomatic carrier cattle was achieved and will allow the analysis of parasite diversity within cattle herds. Babesia divergens / carrier cattle / in vitro culture / isolation

Antibody Prevalence and Molecular Identification of Babesia Spp. In Roe Deer in France

“…Here in 2005, even when almost half of the animals were found to be infected by using cell culture (42/96), Babesia could not be detected by direct examination of Giemsa-stained smears. In domestic ruminants, RBC culture has a detection threshold for B. divergens of about 10 27 % of parasitemia, corresponding to about 10 parasites/ml of blood (Malandrin et al, 2004). There may be persistence of infection even at this low level, as demonstrated in sheep experimentally infected with B. divergens and experiencing a persistent IgG response, despite no currently detectable parasites by RBC culture (Moreau et al, 2009).…”

“…Direct examination was performed under a microscope after Giemsa-based staining of whole blood smears (1003; Diff-Quick, Dade Behring, Deerfield, Illinois, USA). For autologous cultivation (i.e., with red blood cells [RBCs] from the sample), the RBC pellet was washed twice in Roswell Park Memorial Institute (RPMI) 1640 (Lonza, Levallois-Perret, France) (with centrifugation at 800 3 G), and 150 ml was cultivated in 2-ml wells in RPMI supplemented with 20% decomplemented fetal calf serum (Lonza; Malandrin et al, 2004). When parasitemia reached 10 to 20% (after 2-3 wk in autologous cell culture), blood cells were taken for molecular characterization of isolates.…”

Natural Transmission of ZoonoticBabesiaspp. byIxodes ricinusTicks

“…EU1. A dairy farm in La Verrie (Vendée, France) was selected as a favorable biotope for B. divergens transmission on the basis not only of the presence of numerous ticks on cows and in pastures in 2007 but also of the parasite circulation in the herd, attested by serologic testing (prevalence of 37.5% by immunofl uorescence antibody test [IFAT]) and confi rmed by its isolation from cattle erythrocytes (prevalence 25% by culture) (9). Of the cows tested by IFAT, 56% had positive results, which indicated that new infections from ticks were occurring within the herd.…”

In Vitro Cultivation of a ZoonoticBabesiasp. Isolated from Eastern Cottontail Rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts

“…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.…”