Transfection systems for Babesia bovis: A review of methods for the transient and stable expression of exogenous genes

With the recently sequenced Babesia bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock-out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3′ region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1α promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovis ef-1α locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.     

In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive

The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a₁, a₂, b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a₁; YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.     

Babesia bovis contains an abundant parasite-specific protein-free glycerophosphatidylinositol and the genes predicted for its assembly

Autonomous glycosylphosphatidylinositol (GPI) molecules (also protein-free GPIs or free GPIs) have been reported to be particularly abundant in some parasitic protozoa and mediate strong immunomodulatory effects on the host immune system. In the work at hand we have investigated the existence of free GPIs in Babesia bovis. Comparative thin layer chromatographic analysis of the protein-free glycolipid fraction of in vitro cultured B. bovis merozoites and erythrocyte membranes demonstrated the presence of an abundant parasite-specific band. Its chemical analysis revealed a GPI species containing a chain of two mannose residues, N-glucosamine and non-acylated inositol. The lipid moiety linked to inositol was diacylglycerol. The total fatty acid composition showed predominantly long-carbon chain molecules (12% of C_22:0 and 45% of C_24:0). The potential of B. bovis to assemble the presented free GPI species was verified by the existence of seven genes in its genome that putatively encode the following GPI biosynthetic enzymes: PI N-acetyl-GlcN-transferase (PIG-A and GPI-1), N-acetyl-GlcN-PI-de-N-acetylase (PIG-L), acyltransferase (PIG-W), dolichyl-phosphate mannosyl transferase (DPM-1), GPI mannosyltransferase I (PIG-M), and GPI mannosyltransferase II (PIG-V). GPI biosynthesis is vital for the intraerythrocytic parasite stage as mannosamine, an inhibitor of GPI biosynthesis, impaired in vitro growth of B. bovis merozoites. Absence of the vast majority of N-glycan metabolism encoding genes in the B. bovis genome underscores that the growth inhibitory effect of mannosamine is attributable to its interference with GPI biosynthesis and not with assembly of N-linked oligosaccharides, as has been described for higher eukaryotes. Elucidation of the structure and biosynthesis of GPI may allow to facilitate the development of future immune interventions against bovine babesiosis.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

Bovine babesiosis: The immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn B. argentina)

Soluble antigen which protected susceptible cattle against challenge with $\text{Babesia bovis}$ was extracted from $\text{B. bovis}$-infected erythrocytes by sonic disintegration and separation of the soluble from the insoluble matter by ultracentrifugation. The material was then fractionated by the precipitation of fibrinogen-like proteins. The precipitate contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen originally located on the parasite remained in solution. Both fractions conferred protection on splenectomized calves against challenge with $\text{B. bovis}$. However, the fraction containing the parasite antigens appeared to have more potential for development as a killed vaccine because it was not heavily contaminated with antigenic material from bovine erythrocytes.     

<Emphasis Type="Italic">Eimeria bovis</Emphasis> meront I-carrying host cells express parasite-specific antigens on their surface membrane

Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.     

Arginine promotes myogenic differentiation and myotube formation through the elevation of cytoplasmic calcium concentration

This study aimed to explore the mechanism underlying arginine-promoted myogenesis of myoblasts. C2C12 cells were cultured with a medium containing 0.1, 0.4, 0.8, or 1.2 mmol/L arginine, respectively. Cell proliferation, viability, differentiation indexes, cytoplasmic Ca²⁺ concentration, and relative mRNA expression levels of myogenic regulatory factors (MRF) and key Ca²⁺ channels were measured in the absence or presence of 2 chemical inhibitors, dantrolene (DAN, 10 μmol/L) and nisoldipine (NIS, 10 μmol/L), respectively. Results demonstrated that arginine promoted myogenic differentiation and myotube formation. Compared with the control (0.4 mmol/L arginine), 1.2 mmol/L arginine upregulated the relative mRNA expression levels of myogenin (MyoG) and Myomaker at d 2 during myogenic induction (P < 0.05). Cytoplasmic Ca²⁺ concentrations were significantly elevated by arginine supplementation at d 2 and 4 (P < 0.05). Relative mRNA expression levels of Ca²⁺ channels including the type 1 ryanodine receptor (RyR1) and voltage-gated Ca²⁺ channel (Cav1.1) were upregulated by 1.2 mmol/L arginine during 2-d myogenic induction (P < 0.01). However, arginine-promoted myogenic potential of myoblasts was remarkably compromised by DAN and NIS, respectively (P < 0.05). These findings evidenced that the supplementation of arginine promoted myogenic differentiation and myotube formation through increasing cytoplasmic Ca²⁺ concentration from both extracellular and sarcoplasmic reticulum Ca²⁺.     

Studies on synchronous egress of coccidian parasites (<Emphasis Type="Italic">Neospora caninum,Toxoplasma gondii,Eimeria bovis)</Emphasis> from bovine endothelial host cells mediated by calcium ionophore A23187

Neospora caninum, Toxoplasma gondii and Eimeria bovis are coccidian parasites of veterinary importance. Tachyzoites of N. caninum and T. gondii and sporozoites of E. bovis are able to invade and replicate in endothelial cells in vivo and in vitro. As it holds true for all eukaryotic cells, the survival of parasitized host cells and the parasites themselves should be dependent on ion balances, especially on extra- and intracellular calcium concentrations. Addition of the calcium ionophore A23187 reliably did release merozoites from mature N. caninum and T. gondii meronts grown in cultured primary bovine umbilical vein endothelial cells (BUVEC). Extent and time course of merozoite release depended on both, maturity of the meronts and concentration of the calcium ionophore. Attempts, however, to achieve synchronous release of merozoites from E. bovis first generation meronts by ionophore treatment failed, suggesting a different biological behaviour of this parasite. According to microscopical observations, the quite variable time of E. bovis macromeront maturation and a hampered merozoite exit owing to dense parasite-induced cytoskeleton elements surrounding the meront may be a reason for the lack of inducible synchronous release. Electronic supplementary materials The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.     

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.     

Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China

Mycoplasma bovis has spread widely throughout the world via animal movement and has become an important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations of antimicrobials for Mycoplasma bovis have not been studied in China. The objective of this study was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolated from young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45 bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible or had medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant to macrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in the rrnA operon in domain V of 23S rRNA was observed in strains that were resistant to macrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in the resistance of Mycoplasma bovis strains to macrolides.     

Molecular findings and approaches spotlighting Mycobacterium bovis persistence in cattle

Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (M. bovis) are the etiological agents of human and bovine tuberculosis (TB, bTB) respectively, and share genetic identity over 99% at the whole genome level. Progress has been made towards explaining how mycobacteria and their infected hosts remain in balance without producing clinical symptoms of disease, a phenomenon referred to as latency or persistence, which can be mimicked by certain in vitro conditions. Latency/persistence has mainly been studied using Mtb, where the two-component signalling system, dosRS, has been assigned an instrumental role, and even constitutes the current basis for development of new diagnostic methods and treatment addressing this particular stage of TB. M. bovis conserves homolog genes that in Mtb play a role in human latent TB infection and that, by analogy, would allow it to enter a persistent state in infected cattle; nevertheless, little attention has been paid to this stage in bovine hosts. We suggest that many of the advances acquired through the study of Mtb can and should be taken into consideration by research groups and veterinary professionals dealing with bTB. The study of the infection in bovines, paying particular attention to defining the molecular and cellular markers of a M. bovis persistent infection in cattle, presents great opportunities for the development and trial of new diagnostic tests and vaccines, tools that will surely help in promoting eradication of bTB in high-burden settings.     

Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.     

Haemolytic activity of sheep complement for two assay systems

Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with rabbit antibody (sheep E-rabbit A) provided serum is used as soon as possible after collection. If left at 4 °C to separate from the clot, serum C activity for sheep E-rabbit A is markedly reduced. Heparinized plasma retains its haemolytic titre for at least 24 h at 4 °C. Plasma from Mg²⁺-ethyleneglycoltetraacetic acid (EGTA) blood is non-haemolytic, but addition of Ca²⁺ partially restores the titre. A high concentration of rabbit A is necessary to sensitize sheep E.Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg²⁺-EGTA. This C activity is stable at 4 °C for 24 h in serum, Mg²⁺-EGTA plasma and heparinized plasma. Haemolytic activity of serum heated at 50 °C for 30 min was restored by a factor B containing CM-cellulose fraction of foetal lamb serum in the presence of Mg²⁺-EGTA for human E-sheep A but not sheep E-rabbit A.These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca²⁺-Mg²⁺-dependent pathway that is labile in vitro for 24 h at 4 °C.     

Detection of Corynebacterium bovis infection in athymic nude mice from a research animal facility in Korea

Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, this is the first report of C. bovis infection in nude mice from Korea.     

Increased calcium deposits and decreased Ca-ATPase in erythrocytes of ascitic broiler chickens

The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca²⁺ and Ca²⁺-ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca²⁺-ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca²⁺-ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.     

Dynamic analysis of Ca2+ level during bovine oocytes maturation and early embryonic development

Mammalian oocyte maturation and early embryo development processes are Ca²⁺-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca²⁺ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca²⁺ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca²⁺ was present throughout the blastomere. In PA embryos, Ca²⁺ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca²⁺ in the SCNT embryos. However, Ca²⁺ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca²⁺ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca²⁺ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.