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Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

ABSTRACT: BACKGROUND:Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected cattle would affect parasite transmission to the next generation of ticks. RESULTS:Silencing expression of the vitellogenin receptor in the ovary by RNA interference, resulted in reduced tick fertility. We observed reduced egg production (i.e. reduced weight of eggs), a lower rate of embryonic development, and a reduction in hatching. Analysis of individual larvae by PCR confirmed that RNAi mediated downregulation of the R. microplus vitellogenin receptor and also interfered with transovarial transmission of B. bovis. None of the larvae (0/58) from the RmVgR dsRNA-injected group were PCR-positive, whereas 12% (7/58) and 17% (10/58) of larvae from the non-injected and buffer-injected control groups, respectively, were infected with B. bovis. CONCLUSIONS:The combined effects of reduced fecundity and reduced infection in surviving larvae resulting from silencing indicate that vitellogenin receptor is essential for tick reproduction and may play a vital role in B. bovis transmission.

SUBMITTER: Hussein HE

PROVIDER: S-EPMC6321724 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

Hussein Hala E HE Johnson Wendell C WC Taus Naomi S NS Suarez Carlos E CE Scoles Glen A GA Ueti Massaro W MW

Parasites & vectors 20190105 1

<h4>Background</h4>Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected catt ...[more]

PMID: 30611310

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Project description:BACKGROUND: Cattle 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. METHODS: Total 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. RESULTS: Ticks 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. CONCLUSION: Our 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.

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.

Project description:Water buffaloes can be infected by tick-borne pathogens (TBPs) in endemic areas where cattle and buffalo coexist. Among TBPs affecting buffaloes is the Apicomplexan hemoparasites Babesia bovis and B. bigemina, transmitted by Rhipicephalus microplus ticks. However, little empirical evidence exists on whether buffalo can support TBPs' infection and transmission. A cohort study was designed to measure the infestation levels of R. microplus in buffaloes as well as the ability of buffalo-fed ticks to transmit B. bovis and B. bigemina to their offspring. Tick infestation of different life stages was quantified in cattle and buffalo kept in field conditions in western Cuba. Engorged adult female ticks were allowed to lay eggs in controlled conditions of humidity and temperature, and reproductive parameters were measured and analyzed. Hosts and tick larvae were tested for the presence of Babesia spp. using species-specific qPCR assays. Tick infestation was not observed in adult buffaloes. However, buffalo and cattle calves were equally infested, although the larval survival rate was higher in cattle calves than in buffalo calves. All larval pools (31) obtained from the adult female ticks were positive for B. bovis, whereas only 68% (21/31) was positive for B. bigemina. Among the 10 larval pools negative for B. bigemina, three proceeded from adult females fed on Babesia-negative buffaloes. The other seven pools were from Babesia-positive animals, three from cattle and four from buffalo calves. Babesia infection levels in tick larvae, quantified by qPCR, were similar in female ticks fed on buffalo and bovine calves. We conclude that water buffalo can sustain tick vector populations and support Babesia infection in levels high enough as to be infective for ticks. Our results also validated the hypothesis that adult female ticks fed on buffalo can transmit the pathogens B. bovis and B. bigemina to their offspring. Nevertheless, further laboratory studies are needed to address the question of whether the transovarial transmission of Babesia occurs in the following settings: (1) When adult females are infected previous to the feeding on the buffalo or/and (2) when the adult females acquire the infection while feeding on the buffalo.

Dataset Information

Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

Publications

Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

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