A differential diagnostic criterion for Babesia major and Babesia bigemina vermicules from tick haemolymph

Summary Babesia major mature and immature vermicules in the haemolymph of Haemaphysalis punctata were measured and found to be significantly larger than vermicules of Babesia bigemina. Mature B. major vermicules had a mean length of 15.53 m and mature B. bigemina vermicules had a mean length of 11.79 m. This difference provides a new criterion for the differentiation of the two species.     

Detection of tick blood parasites in Egypt using PCR assay I—Babesia bovis and Babesia bigemina

Babesia bovis and Babesia bigemina are distributed all over the world; the etiologic agents of the animal babesiosis are considered the most important tick-borne disease. The present research work was the first attempt to determine the prevalence of B. bovis and B. bigemina infection in ticks, in Egypt, by using polymerase chain reaction (PCR). Questing 5,243 hard and soft ticks were collected from different localities throughout the Giza Governorate. Furthermore, DNA from 500 different individual tick species was extracted and PCR was performed. Primers verified from the sequence of Mexico strain of both species were used. Two fragments of 275 and 175 bp of B. bovis and B. bigemina, respectively, were generated. Fragments of the pathogens were recovered with PCR and sequenced. The prevalence of B. bovis and B. bigemina in Boophilus annulatus ticks were 55% and 66%, respectively. Also, presence of 12% dual infection with B. bovis and B. bigemina was observed. Sequence analysis of PCR product of these pathogens shares a high degree of similarity in sequence compared to similar species found in GenBank.     

The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis and its potential application as a bioacaricide for tick control

Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (>30°C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p<0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.     

Characterization and antiviral activity of a newly identified defensin-like peptide,HEdefensin, in the hard tick Haemaphysalis longicornis

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.     

A novel C-type lectin with triple carbohydrate recognition domains has critical roles for the hard tick Haemaphysalis longicornis against Gram-negative bacteria

C-type lectins (CLecs) play an important role in innate immunity against invaders. In this study, a novel CLec was identified from Haemaphysalis longicornis ticks (HlCLec). HlCLec contains a signal peptide and a transmembrane region. Interestingly, HlCLec possesses three dissimilar carbohydrate recognition domains (CRDs). Each CRD contains the mutated motif of Ca²⁺-binding site 2. HlCLec mRNA was up-regulated during blood feeding, and had highest expression in the midgut and ovary. HlCLec localization was also confirmed by immunofluorescent antibody test (IFAT). HlCLec was found on the cell membrane and basal lamina of midgut and ovary. In addition, the recombinant HlCLec and individual CRDs demonstrated direct binding activity to Escherichia coli and Staphylococcus aureus; however, no growth inhibition activity was observed. Furthermore, E. coli injection after silencing of HlCLec caused drastic reduction in survival rate of ticks. These results strongly suggest the key role of HlCLec in tick innate immunity against Gram-negative bacteria.     

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21?% for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5?% samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5?%, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2?% in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3?% in Chiang Rai, 1.9?% in Chiang Mai, and 13.7?% in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.     

Bovine babesiosis: induction of protective immunity with culture-derived Babesia bovis and Babesia bigemina immunogens

The comparative protective efficacy of culture-derived Babesia bovis and B. bigemina immunogens against challenge exposure of susceptible crossbred cattle to heterologous strains was examined and correlated with the antigenic threshold requirements for induction of protective immunity. Strong protection was observed in animals vaccinated with 10 ml-equivalent doses of soluble, B. bovis exoantigen-containing supernatant fluids. Similar protective responses to B. bigemina exoantigens were evident even at 1 ml-equivalent dosages. In addition, the efficacy of a combined B. bovis-B. bigemina immunogen was assessed with a dose-response analysis in highly susceptible, purebred cattle. Vaccinated animals were protected against clinical babesiosis, and significant weight gains were recorded after challenge infection with virulent parasites.     

A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle

In this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.     

The immunosuppressive functions of two novel tick serpins,HlSerpin-a and HlSerpin-b,from Haemaphysalis longicornis

Serpins are evolutionarily conserved serine protease inhibitors that are widely distributed in animals, plants and microbes. In this study, we reported the cloning and functional characterizations of two novel serpin genes, HlSerpin-a and HlSerpin-b, from the hard tick Haemaphysalis longicornis of China. Recombinant HlSerpin-a and HlSerpin-b displayed protease inhibitory activities against multiple mammalian proteases. Similar to other tick serpins, HlSerpin-a and HlSerpin-b suppressed the expression of inflammatory cytokines such as TNF-α, interleukin (IL)-6 and IL-1β from lipopolysaccharide-stimulated mouse bone-marrow-derived macrophages (BMDMs) or mouse bone-marrow-derived dendritic cells (BMDCs). The minimum active region (reaction centre loop) of HlSerpin-a, named SA-RCL, showed similar biological activities as HlSerpin-a in the protease inhibition and immune suppression assays. The immunosuppressive activities of full-length HlSerpin-a and SA-RCL are impaired in Cathepsin G or Cathepsin B knockout mouse macrophages, suggesting that the immunomodulation functions of SA and SA-RCL are dependent on their protease inhibitory activity. Finally, we showed that both full-length HlSerpins and SA-RCL can relieve the joint swelling and inflammatory response in collagen-induced mouse arthritis models. These results suggested that HlSerpin-a and HlSerpin-b are two functional arthropod serpins, and the minimal reactive peptide SA-RCL is a potential candidate for drug development against inflammatory diseases.     

Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining

Summary The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.     

Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29-kilodalton protein and its effect as a vaccine against tick infestation in rabbits

The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized a Haemaphysalis longicornis tick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and mature H. longicornis ticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.     

The infection of various tick species with Babesia bigemina,its transmission and identification

Strains of Boophilus decoloratus and B. microplus were easily infected with a single stock of Babesia bigemina; Boophilus annulatus could be infected less easily, and it was difficult to infect Rhipicephalus evertsi. Two strains of R. appendiculatus and one strain each of R. bursa and Hyalomma anatolicum excavatum were refractory. The same stock of B. bigemina was transmitted by nymphs and adults of B. decoloratus but only by nymphs of R. evertsi. Vertical infection was not observed in R. evertsi, whereas it persisted in B. decoloratus for at least two generations. The sporokinetes in the hemolymph of R. evertsi were significantly shorter than those in the three Boophilus species.     

Inhibitory effect of allicin on the growth of Babesia and Theileria equi parasites

Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC₅₀ values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P?<?0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P?<?0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.