Surveillance of avian malaria and related haemoparasites in common terns (Sterna hirundo) on the Atlantic coast of South America.

Haemosporidia (Apicomplexa, Haemosporida) are protozoa that infect vertebrate blood cells and are transmitted by vectors. Among vertebrates, birds possess the greatest diversity of haemosporidia, historically placed in 3 genera: Haemoproteus, Leucocytozoon and Plasmodium, the causative agent of avian malaria. In South America, existing data on haemosporidia are spatially and temporally dispersed, so increased surveillance is needed to improve the determination and diagnosis of these parasites. During the non-breeding season in 2020 and 2021, 60 common terns (Sterna hirundo) were captured and bled as part of ongoing research on the population health of migratory birds on the Argentinian Atlantic coast. Blood samples and blood smears were obtained. Fifty-eight samples were screened for Plasmodium, Haemoproteus and Leucocytozoon, as well as for Babesia parasites by nested polymerase chain reaction and by microscopic examination of smears. Two positive samples for Plasmodium were found. The cytochrome b lineages detected in the present study are found for the first time, and are close to Plasmodium lineages found in other bird orders. The low prevalence (3.6%) of haemoparasites found in this research was similar to those reported for previous studies on seabirds, including Charadriiformes. Our findings provide new information about the distribution and prevalence of haemosporidian parasites from charadriiforms in the southernmost part of South America, which remains understudied.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Isolation and characterization of Babesia pecorum sp. nov. from farmed red deer (Cervus elaphus).

The diversity of Babesia species infecting cervids in parts of central and southern Spain was analyzed by collecting blood from farmed red deer (Cervus elaphus). Babesia sp. was isolated in vitro from two red deer herds in Cádiz and Ciudad Real. The number of Babesia sp. carriers differed between the two herds: 36/77 in Cádiz and 1/35 in Ciudad Real. Hyalomma lusitanicum was the most prevalent tick species identified on the Cádiz farm vegetation and on sampled animals, and is therefore a candidate vector. The molecular characteristics of 21 isolates were determined by complete (8 isolates) or partial (13 isolates) 18S rRNA gene sequencing. The sequences were highly similar (over 99.4% identity) and 6 sequence types were identified at the level of one herd only, demonstrating a rather high genetic diversity. They formed a monophyletic clade, and members of the three main sequence types shared a similar morphology and the same erythrocyte susceptibility pattern. This clade also included Babesia sp. Xinjiang isolated from sheep in China and Babesia sp. identified in giraffe in South Africa, with identities higher than 98.3% and statistically relevant phylogenetic support. None of the biological properties analyzed for both Babesia from red deer and Babesia sp. Xinjiang allowed their differentiation (ability to develop in vitro in erythrocytes from cattle and sheep, as well as in erythrocytes from different cervids, unsuccessful infection of calves). We propose the Babesia isolated from red deer as a new species named B. pecorum. Whether Babesia sp. Xinjiang and the Babesia characterized in South Africa belong to the same species is debated.

Spatial patterns of Hyalomma marginatum-borne pathogens in the Occitanie region (France), a focus on the intriguing dynamics of Rickettsia aeschlimannii.

Hyalomma marginatum is an invasive tick species recently established in mainland southern France. This tick is known to host a diverse range of human and animal pathogens. While information about the dynamics of these pathogens is crucial to assess disease risk and develop effective monitoring strategies, few data on the spatial dynamics of these pathogens are currently available. We collected ticks in 27 sites in the Occitanie region to characterize spatial patterns of H. marginatum-borne pathogens. Several pathogens have been detected: Theileria equi (9.2%), Theileria orientalis (0.2%), Anaplasma phagocytophilum (1.6%), Anaplasma marginale (0.8%), and Rickettsia aeschlimannii (87.3%). Interestingly, we found a spatial clustered distribution for the pathogen R. aeschlimannii between two geographically isolated areas with infection rates and bacterial loads significantly lower in Hérault/Gard departments (infection rate 78.6% in average) compared to Aude/Pyrénées-Orientales departments (infection rate 92.3% in average). At a smaller scale, R. aeschlimannii infection rates varied from one site to another, ranging from 29% to 100%. Overall, such high infection rates (87.3% on average) and the effective maternal transmission of R. aeschlimannii might suggest a role as a tick symbiont in H. marginatum. Further studies are thus needed to understand both the status and the role of R. aeschlimannii in H. marginatum ticks.IMPORTANCETicks are obligatory hematophagous arthropods that transmit pathogens of medical and veterinary importance. Pathogen infections cause serious health issues in humans and considerable economic loss in domestic animals. Information about the presence of pathogens in ticks and their dynamics is crucial to assess disease risk for public and animal health. Analyzing tick-borne pathogens in ticks collected in 27 sites in the Occitanie region, our results highlight clear spatial patterns in the Hyalomma marginatum-borne pathogen distribution and strengthen the postulate that it is essential to develop effective monitoring strategies and consider the spatial scale to better characterize the circulation of tick-borne pathogens.

Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks.

Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 µm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

Prevalence of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Rickettsia spp. and Babesia spp. in cattle serum and questing ticks from Belgium.

BACKGROUND: Anaplasmosis, borreliosis, rickettsiosis and babesiosis are tick-borne diseases of medical, veterinary and economic importance. In Belgium, little is known on the prevalence of these diseases in animals and previous screenings relate only to targeted geographic regions, clinical cases or a limited number of tested samples. We therefore performed the first nationwide seroprevalence study of Anaplasma spp., A. phagocytophilum, Borrelia spp., Rickettsia spp. and Babesia spp. in Belgian cattle. We also screened questing ticks for the aforementioned pathogens. METHODS: ELISAs and IFATs were performed on a representative sample set of cattle sera stratified proportionally to the number of cattle herds per province. Questing ticks were collected in areas where the highest prevalence for the forenamed pathogens in cattle serum were observed. Ticks were analyzed by quantitative PCR for A. phagocytophilum (n = 783), B. burgdorferi sensu lato (n = 783) and Rickettsia spp. (n = 715) and by PCR for Babesia spp. (n = 358). RESULTS: The ELISA screening for antibodies to Anaplasma spp. and Borrelia spp. in cattle sera showed an overall seroprevalence of 15.6% (53/339) and 12.9% (52/402), respectively. The IFAT screening for antibodies against A. phagocytophilum, Rickettsia spp. and Babesia spp. resulted in an overall seroprevalence of 34.2% (116/339), 31.2% (99/317) and 3.4% (14/412), respectively. At the provincial level, the provinces of Liege and Walloon Brabant harboured the highest seroprevalence of Anaplasma spp. (44.4% and 42.7% respectively) and A. phagocytophilum (55.6% and 71.4%). East Flanders and Luxembourg exhibited the highest seroprevalence of Borrelia spp. (32.4%) and Rickettsia spp. (54.8%) respectively. The province of Antwerp showed the highest seroprevalence of Babesia spp. (11%). The screening of field-collected ticks resulted in a prevalence of 13.8% for B. burgdorferi s.l., with B. afzelii and B. garinii being the most common genospecies (65.7% and 17.1%, respectively). Rickettsia spp. was detected in 7.1% of the tested ticks and the only identified species was R. helvetica. A low prevalence was found for A. phagocytophilum (0.5%) and no Babesia positive tick was detected. CONCLUSIONS: The seroprevalence data in cattle indicate hot spots for tick-borne pathogens in specific provinces and highlights the importance of veterinary surveillance in anticipating the emergence of diseases among humans. The detection of all pathogens, with the exception of Babesia spp. in questing ticks, underlines the need of raising awareness among public and professionals on other tick-borne diseases along with lyme borreliosis.

Babesia bennetti: A chimeric sequence for a true parasite?

In this Letter, the possibility is raised and evidenced that the published sequence of the avian piroplasm Babesia bennetti is a chimera. This is an important taxonomic and phylogenetic issue, as this unique sequence creates a second clade of avian piroplasms within Babesia sensu stricto.

Characterization and diversity of Babesia sp. YLG, a new member of the Peircei group infecting Mediterranean yellow-legged gulls (Larus michahellis).

Avian infecting piroplasms are largely under-studied compared to other hemoparasites, and this paucity of information has blurred our phylogenetic and biological comprehension of this important group as a whole. In the present study, we detected and characterized Babesia from yellow-legged gull (Larus michahellis) chicks from a colony in southern France. Based on morphological and molecular characterizations, a new Babesia species belonging to the Peircei group, a clade of avian-specific piroplasms, was identified. Due to the complexity of species delineations and the low number of parasites characterized in this clade to date, a species name was not yet attributed; we refer to it for now as Babesia sp. YLG (Yellow-Legged Gull). High prevalence (85% and 58% in 2019 and 2020, respectively) and high parasitemia (up to 20% of parasitized erythrocytes) were recorded in chicks, without any obvious clinical signs of infection. Although the 16 isolates examined had identical 18S rRNA gene sequences, six genetic variants were described based on partial cox1 sequencing, with evidence of chicks co-infected by two variants. Transmission of Babesia sp. YLG via the soft tick Ornithodoros maritimus is discussed.

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay.

Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

The New Human Babesia sp. FR1 Is a European Member of the Babesia sp. MO1 Clade.

In Europe, Babesia divergens is responsible for most of the severe cases of human babesiosis. In the present study, we describe a case of babesiosis in a splenectomized patient in France and report a detailed molecular characterization of the etiological agent, named Babesia sp. FR1, as well as of closely related Babesia divergens, Babesia capreoli and Babesia sp. MO1-like parasites. The analysis of the conserved 18S rRNA gene was supplemented with the analysis of more discriminant markers involved in the red blood cell invasion process: rap-1a (rhoptry-associated-protein 1) and ama-1 (apical-membrane-antigen 1). The rap-1a and ama-1 phylogenetic analyses were congruent, placing Babesia sp. FR1, the new European etiological agent, in the American cluster of Babesia sp. MO1-like parasites. Based on two additional markers, our analysis confirms the clear separation of B. divergens and B. capreoli. Babesia sp. MO1-like parasites should also be considered as a separate species, with the rabbit as its natural host, differing from those of B. divergens (cattle) and B. capreoli (roe deer). The natural host of Babesia sp. FR1 remains to be discovered.

Natural transmission of Zoonotic Babesia spp. by Ixodes ricinus ticks.

To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus.

First detection and molecular identification of the zoonotic Anaplasma capra in deer in France.

Cervids are known to be reservoirs of zoonotic bacteria transmitted by ticks. This study aimed to identify the Anaplasma species carried by captive red deer and swamp deer in a wild fauna reserve in France. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR targeting the 23S rRNA was performed to detect and characterize Anaplasma spp. and determine the presence of zoonotic species. Anaplasma phagocytophilum was identified in 14/59 red deer (23.7%) but it was not identified in any of the swamp deer (7 animals). Three sequences could not be assigned to any particular species based on the 23S rRNA sequences. Complementary nested PCR targeting 16S rRNA, gltA and groEL genes and sequencing analysis then identified these sequences as a recently reported zoonotic species, Anaplasma capra; this species was found in 2 red deer (Cervus elaphus) and 1 swamp deer (Rucervus duvaucelii). This is the first report of the tick-borne zoonotic bacterium A. capra in France, a species otherwise described only in China, Japan, Malaysia and South Korea in goats, sheep, deer, cattle and Japanese serows (Capricornis crispus). While this bacterium may have been introduced into the reserve by infected imported animals, its local epidemiological cycle via tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.

Cell cycle characteristics of crenarchaeota: unity among diversity.

The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.

The Complexity of Piroplasms Life Cycles.

Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of Babesia, Theileria, and Cytauxzoon representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of Babesia or Theileria tick vectors.

Infection Kinetics and Tropism of Borrelia burgdorferi sensu lato in Mouse After Natural (via Ticks) or Artificial (Needle) Infection Depends on the Bacterial Strain.

Borrelia burgdorferi sl is a complex of pathogen bacteria transmitted to the host by Ixodes ticks. European Ixodes ricinus ticks transmit different B. burgdorferi species, pathogenic to human. Bacteria are principally present in unfed tick midgut, then migrate to salivary glands during blood meal and infect a new host via saliva. In this study, efficiency of transmission in a mouse model of three pathogen species belonging to the B. burgdorferi sl complex, B. burgdorferi sensu stricto (B31, N40, and BRE-13), B. afzelii (IBS-5), and B. bavariensis (PBi) is examined in order to evaluate infection risk after tick bite. We compared the dissemination of the Borrelia species in mice after tick bite and needle injection. Location in the ticks and transmission to mice were also determined for the three species by following infection kinetics. After inoculation, we found a significant prevalence in the brain for PBi and BRE-13, in the heart, for PBi, in the skin where B31 was more prevalent than PBi and in the ankle where both B31 and N40 were more present than PBi. After tick bite, statistical analyses showed that BRE-13 was more prevalent than N40 in the brain, in the bladder and in the inguinal lymph node. When Borrelia dissemination was compared after inoculation and tick bite, we observed heart infection only after tick inoculation of BRE-13, and PBi was only detected after tick bite in the skin. For N40, a higher number of positive organs was found after inoculation compared to tick bite. All European B. burgdorferi sl strains studied were detected in female salivary glands before blood meal and infected mice within 24 h of tick bite. Moreover, Borrelia-infected nymphs were able to infect mice as early as 12 h of tick attachment. Our study shows the need to remove ticks as early as possible after attachment. Moreover, Borrelia tropism varied according to the strain as well as between ticks bite and needle inoculation, confirming the association between some strains and clinical manifestation of Lyme borreliosis, as well as the role played by tick saliva in the efficiency of Borrelia infection and dissemination in vertebrates.

Detecting and characterizing mixed infections with genetic variants of Anaplasma phagocytophilum in roe deer (Capreolus capreolus) by developing an ankA cluster-specific nested PCR.

BACKGROUND: Anaplasma phagocytophilum is a tick-transmitted Gram-negative obligate intracellular bacterium able to infect a wide variety of wild and domestic animals worldwide. Based on the genetic diversity observed with different molecular markers, several host-specific lineages have been identified. Roe deer is one of the most important reservoirs of this bacterium and hosts different genetic groups sometimes found on domestic animals. We therefore developed an ankA cluster-specific nested PCR (nPCR) to evaluate the prevalence of the three different ankA genetic groups described in roe deer (clusters II, III and IV) at three locations in France and the level of co-infections. RESULTS: The specificity of the three nPCRs was assessed by partially sequencing 35 amplicons of ankA genes obtained from the different nested PCRs. All three genetic lineages were detected in roe deer from all three geographical locations. Of the infected deer population, 60.7% were co-infected by two or three different genetic variants. Co-infections varied from 42.9 to 70.6% of the infected population depending on the local infection prevalences (from 33.3 to 73.9%). All types of mixed infections occurred, suggesting the absence of a strict variant exclusion by another variant. CONCLUSIONS: Mixed infections by two or three genetic variants of A. phagocytopilum are a common feature in roe deer. Genetic variants (cluster IV) also found in domestic ruminants (cattle and sheep) were present in all the roe deer populations analyzed, suggesting a shared epidemiological cycle.

Stimulation and quantification of Babesia divergens gametocytogenesis.

BACKGROUND: Babesia divergens is the most common blood parasite in Europe causing babesiosis, a tick-borne malaria-like disease. Despite an increasing focus on B. divergens, especially regarding veterinary and human medicine, the sexual development of Babesia is poorly understood. Development of Babesia sexual stages in the host blood (gametocytes) plays a decisive role in parasite acquisition by the tick vector. However, the exact mechanism of gametocytogenesis is still unexplained. METHODS: Babesia divergens gametocytes are characterized by expression of bdccp1, bdccp2 and bdccp3 genes. Using previously described sequences of bdccp1, bdccp2 and bdccp3, we have established a quantitative real-time PCR (qRT-PCR) assay for detection and assessment of the efficiency of B. divergens gametocytes production in bovine blood. We analysed fluctuations in expression of bdccp genes during cultivation in vitro, as well as in cultures treated with different drugs and stimuli. RESULTS: We demonstrated that all B. divergens clonal lines tested, originally derived from naturally infected cows, exhibited sexual stages. Furthermore, sexual commitment was stimulated during continuous growth of the cultures, by addition of specific stress-inducing drugs or by alternating cultivation conditions. Expression of bdccp genes was greatly reduced or even lost after long-term cultivation, suggesting possible problems in the artificial infections of ticks in feeding assays in vitro. CONCLUSIONS: Our research provides insight into sexual development of B. divergens and may facilitate the development of transmission models in vitro, enabling a more detailed understanding of Babesia-tick interactions.

RAP-1a is the main rhoptry-associated-protein-1 (RAP-1) recognized during infection with Babesia sp. BQ1 (Lintan) (B. motasi-like phylogenetic group), a pathogen of sheep in China.

Babesia sp. BQ1 (Lintan) is one of the parasites isolated from infected sheep in China that belongs to the B. motasi-like phylogenetic group. The rhoptry-associated-protein 1 (rap-1) locus in this group consists of a complex organization of 12 genes of three main types: 6 rap-1a variants intercalated with 5 identical copies of rap-1b and a single 3' ending rap-1c gene. In the present study, transcription analysis performed by standard RT-PCR demonstrated that the three different rap-1 gene types and the four rap-1a variants were transcribed by the parasite cultivated in vitro. Peptides, specific for each rap-1 type gene, were selected in putative linear B-epitopes and used to raise polyclonal rabbit antisera. Using these sera, the same expression pattern of RAP-1 proteins was found in parasites cultivated in vitro or collected from acute infection whereas only RAP-1a67 was detectable in merozoite extracts. However, ELISA performed with recombinant RAP-1a67, RAP-1b or RAP-1c and sera from infected sheep demonstrated that RAP-1a67 is the main RAP-1 recognized during infection, even if some infected sheep also recognized RAP-1b and/or RAP-1c.

Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and ß. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two ß genes was demonstrated by standard RT-PCR.

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro.

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.