The currently accepted initiation of Babesia infection describes a sporozoite stage infused into the host, along with other saliva components, by the tick vector. This sporozoite can enter and initiate erythrocyte infection directly. In the particular case of Babesia microti , however, that sporozoite loses the ability to further propagate in vitro once deprived of its natural host. True B. sensu stricto do not require the host collaboration described in this study. Hence it has become a current topic of research involving B. microti ( B. sensu lato ), a rather unique species that requires host collaboration to maintain an erythrocyte propagation cycle. The main attachment protein is synthesized by this parasite in excess and exported to the host from the erythrocyte infrastructure to immunize the host at all stages of infection. The synthesis of host immune IgM antibody is necessary for the propagation of B. microti , being central to entry into uninfected host erythrocytes. Sequential use of the host immune system then involves complement factor C3b to complete the three-part assembly necessary to initiate the rhoptry sequence for invasion of uninfected erythrocytes and further propagation. These several components must be furnished within the in vitro culture medium and the sequence of these reactions is discussed. The corollary view of the parasite survival versus the host immune defenses is also discussed as it involves the same host factors promoting continuing parasite growth. This is the first description of continuous in vitro propagation of B. microti .

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5′ putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

For many tick-borne organisms, dogs may be utilized as sentinel hosts to estimate the risk of human infection. The detection of antibodies in these animals indicates the circulation of pathogens in each location for a set period of time. The standard protocol for the surveillance of tick-borne diseases established by the Brazilian National Reference Laboratory for Rickettsiosis at the Ezequiel Dias Foundation (FUNED) includes testing the seropositivity of dogs for Rickettsia spp. and Ehrlichia canis. Dog serum samples were collected during FUNED’s surveillance protocol in a preserved area in Brazil. Despite Anaplasma phagocytophilum being endemic in Brazil, this agent is not included in FUNED’s standard protocol. To determine if A. phagocytophilum should be included in the standard testing protocol, a collaboration with Fuller Laboratories was established and the seropositivity for A. phagocytophilum was tested. The estimated prevalence for singular infections: 25% Rickettsia spp.; 52.3% E. canis; and 70.5% A. phagocytophilum. The estimated prevalence for co-infections: 17% Rickettsia spp. and E. canis; 18.2% Rickettsia spp. and A. phagocytophilum; 39.8% E. canis and A. phagocytophilum; and 13.6% Rickettsia spp., E. canis, and A. phagocytophilum. These results confirmed a significant presence of A. phagocytophilum and establishes a potential threat upon the public health.

Spotted Fever Group (SFG) Rickettsia species maintain an s-layer with two major outer membrane proteins (OmpA and OmpB) aligned within a lipopolysaccharide (LPS) matrix external to the bacterial outer membrane. While the two typhus group species (R. typhi and R. prowazekii) maintain a similar s-layer containing only OmpB with its own specific form of LPS. A major component of these two types of OmpB is a specific and highly conserved beta peptide (β-peptide) non-covalently attached to the respective OmpB passenger domain. This β-peptide is initially translated as the c-terminus of the 168 kDa polypeptide and initially forms a membrane pore to allow the larger passenger domain to exit the bacterial cytoplasm through the outer membrane. Prior to the full exit of this passenger domain from the cytoplasm the long polypeptide chain is cleaved by a peptidase, leaving the c-terminus remaining as a beta-folded membrane pore and the N-terminal passenger domain exiting to the space between the bacterial membrane and the s-layer. The fate of this pore structure and the attachment of β-peptide to the OmpB continues to be examined and will be discussed as another protective component of the Rickettsia. An EIA IgM assay has been developed to utilize this antigen for clinical diagnostic use to accurately detect acute rickettsial infection in testing labs and lead to the availability of rapid test (lateral flow) formats for more immediate testing.