Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine.
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ABSTRACT:
SUBMITTER: Mazuz ML
PROVIDER: S-EPMC7911397 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The protozoan parasite Babesia bovis, a reemerging threat to U.S. cattle, is acquired by adult female ticks of the subgenus Boophilus and is transovarially transmitted as the kinete stage to developing larval offspring. Sporozoites develop within larvae and are transmitted during larval feeding on a bovine host. This study evaluated the efficiency of B. bovis infection within Rhipicephalus (Boophilus) microplus following acquisition feeding on acutely parasitemic cattle. Parasite levels were quantified in blood from experimentally infected cattle and within hemolymph and larvae derived from acquisition-fed female B. microplus. There was a positive correlation between blood parasite levels in acutely parasitemic cattle and kinete levels in the hemolymph of adult female Boophilus ticks following acquisition feeding; however, there was no relationship between kinete levels in females and infection rates of larval progeny. Boophilus microplus females that acquisition fed produced larval progeny with infection rates of 12% to 48%. Importantly, larvae derived from replete females with very low levels of kinete infection, as demonstrated by microscopy and PCR, had infection rates of 22% to 30% and transmitted B. bovis during transmission feeding. These data demonstrate that although hemolymph infection may be undetectable, transmission to larval progeny occurs at a level which ensures transmission to the bovine host.
| S-EPMC1829031 | biostudies-literature
Project description:Babesia bovis is a deadly disease of cattle resulting in severe economic losses in the vast regions of the world where it is endemic. If reintroduced into the United States, babesiosis would cause significant mortality in the naïve cattle population. In order to address the risk to U.S. cattle, it is essential to quantify the transovarial transmission efficiency in adult female Boophilus microplus ticks following acquisition feeding on persistently infected cattle. This study tested the hypothesis that infection rates are the same for larval progeny derived from females fed to repletion during persistent or acute infection. Increasing parasite levels during acute infection correlated with an increasing number of females harboring kinetes detectable in hemolymph (r = 0.9). The percent infected larvae ranged from 0 to 20% when derived from females fed to repletion on persistently infected calves and from 4 to 6% when derived from females fed to repletion during acute parasitemia. There was no significant difference in infection rates of larval progeny, implying that the risk associated with the introduction of either persistently infected or acutely infected cattle is equal. Parasite levels ranged from 2.4 x 10(2) to 1.9 x 10(5) in 3-day-fed larvae derived from females fed to repletion on persistently infected cattle. One group of larvae failed to transmit the parasite, suggesting that a threshold level of parasites must be obtained by larval progeny via transovarial transmission in order for larvae to deliver sufficient parasites to infect a naïve host.
| S-EPMC2045367 | biostudies-literature
Project description:BackgroundCattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world's tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis.MethodsTotal RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR.ResultsTicks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf.ConclusionOur experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis. Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.
| S-EPMC3436708 | biostudies-literature
Project description:BACKGROUND: Rhipicephalus (Boophilus) microplus is an economically important tick of cattle involved in the transmission of Babesia bovis, the etiological agent of bovine babesiosis. Commercial anti-tick vaccines based on the R. microplus Bm86 glycoprotein have shown some effect in controlling tick infestation; however their efficacy as a stand-alone solution for tick control has been questioned. Understanding the role of the Bm86 gene product in tick biology is critical to identifying additional methods to utilize Bm86 to reduce R. microplus infestation and babesia transmission. Additionally, the role played by Bm86 in R. microplus fitness during B. bovis infection is unknown. RESULTS: Here we describe in two independent experiments that RNA interference-mediated silencing of Bm86 decreased the fitness of R. microplus females fed on cattle during acute B. bovis infection. Notably, Bm86 silencing decreased the number and survival of engorged females, and decreased the weight of egg masses. However, gene silencing had no significant effect on the efficiency of transovarial transmission of B. bovis from surviving female ticks to their larval offspring. The results also show that Bm86 is expressed, in addition to gut cells, in larvae, nymphs, adult males and ovaries of partially engorged adult R. microplus females, and its expression was significantly down-regulated in ovaries of ticks fed on B. bovis-infected cattle. CONCLUSION: The R. microplus Bm86 gene plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. Therefore, the data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation particularly in B. bovis endemic areas.
| S-EPMC2994843 | biostudies-literature
Project description:BACKGROUND: Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. METHODS: A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. RESULTS: The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. CONCLUSION: Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.
| S-EPMC4028808 | biostudies-literature
Project description:The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1? (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1? locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1? locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.
| S-EPMC5135042 | biostudies-literature
Project description:Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.
| S-EPMC5036836 | biostudies-literature
Project description:Tick gut glycoprotein, designated as Bm86, found on the luminal surface of the plasma membrane of gut epithelial cells of Boophilus microplus, which is a concealed antigen, has been used as vaccine candidate molecule for immunization against ticks. To better understand the molecular diversity of Bm86 gene in ticks, a portion of the cDNA was sequenced from an Indian isolate of B. microplus. Comparison of nucleotide sequence revealed that Indian isolate had 97 % homology (18 polymorphisms) with that of the Australian isolate and 96 % homology (20 polymorphisms) with that of the Cuban vaccine strain. Further, the Indian isolate differed from the Cuban vaccine isolate at 7 amino acid loci, including 5 substitutions (at residues 88, 94, 175, 176 and 177) and 2 deletions (at 183 and 184). However, protein prediction studies did not show any difference in the putative antigenic epitopes of the protein expressed.
| S-EPMC4087308 | biostudies-literature
Project description:BACKGROUND: The cattle tick Rhipicephalus (Boophilus) microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species. RESULTS: In this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp) in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi) gene and a putative lipocalin (Lpc) gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny. CONCLUSION: The silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.
| S-EPMC2785768 | biostudies-literature
Project description:Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia, including B. bovis. This protozoan parasite has a complex life cycle involving dynamic changes to its transcriptome during the transition between the invertebrate and vertebrate hosts. Studying the role of genes upregulated by tick stage parasites has been hindered by the lack of appropriate tools to study parasite gene products in the invertebrate host. Herein, we present tfBbo5480, a transfected B. bovis cell line, constitutively expressing enhanced green fluorescent protein (eGFP) created by a whole gene replacement transfection strategy, that was capable of completing the parasite's entire life cycle in both the vertebrate and invertebrate hosts. tfBbo5480 was demonstrated to respond to in vitro sexual stage induction and upon acquisition by the female tick vector, Rhipicephalus microplus, the tick specific kinete stage of tfBbo5480 was detected in tick hemolymph. Larvae from tfBbo5480 exposed R. microplus female ticks successfully transmitted the transfected parasite to a naïve calf. The development of the whole gene replacement strategy will permit a deeper understanding of the biology of parasite-host-vector triad interactions and facilitate the evaluation of upregulated genes during the parasite's journey through the tick vector leading to new intervention strategies for the control of bovine babesiosis.
| S-EPMC9229605 | biostudies-literature