Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens.

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm ß-Strand Domain 1 (PißS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PißS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (TFH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

Interactions between Babesia microti merozoites and rat kidney cells in a short-term in vitro culture and animal model.

Babesiosis is one of the most common infections in free-living animals and is rapidly becoming significant among human zoonoses. Cases of acute renal failure in humans caused by Babesia spp. have been described in the literature. The kidneys are characterised by intense blood flow through the blood vessels, which increases the likelihood of contact with the intra-erythrocyte parasite. The aim of this study was to observe the influence of B. microti (ATCC 30221) on renal epithelial cells in vitro cultured (NRK-52E line) and Wistar rats' kidney. Both NRK-52E cells and rats' kidney sections were analysed by light microscopy, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Necrotic changes in renal epithelial cells have been observed in vitro and in vivo. In many cross-sections through the rats' kidney, adhesion of blood cells to the vascular endothelium, accumulation of erythrocytes and emboli were demonstrated. In NRK-52E culture, elements with a distinctly doubled cell membrane resembling B. microti were found inside the cytoplasm and adjacent to the cell layer. The study indicates a chemotactic tendency for B. microti to adhere to the renal tubules' epithelium, a possibility of piroplasms entering the renal epithelial cells, their proliferation within the cytoplasm and emboli formation.

Citizen Science Provides an Efficient Method for Broad-Scale Tick-Borne Pathogen Surveillance of Ixodes pacificus and Ixodes scapularis across the United States.

Tick-borne diseases have expanded over the last 2 decades as a result of shifts in tick and pathogen distributions. These shifts have significantly increased the need for accurate portrayal of real-time pathogen distributions and prevalence in hopes of stemming increases in human morbidity. Traditionally, pathogen distribution and prevalence have been monitored through case reports or scientific collections of ticks or reservoir hosts, both of which have challenges that impact the extent, availability, and accuracy of these data. Citizen science tick collections and testing campaigns supplement these data and provide timely estimates of pathogen prevalence and distributions to help characterize and understand tick-borne disease threats to communities. We utilized our national citizen science tick collection and testing program to describe the distribution and prevalence of four Ixodes-borne pathogens, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti, across the continental United States. IMPORTANCE In the 21st century, zoonotic pathogens continue to emerge, while previously discovered pathogens continue to have changes within their distribution and prevalence. Monitoring these pathogens is resource intensive, requiring both field and laboratory support; thus, data sets are often limited within their spatial and temporal extents. Citizen science collections provide a method to harness the general public to collect samples, enabling real-time monitoring of pathogen distribution and prevalence.

Response of Leucine-Rich Repeat Domain-Containing Protein in Haemaphysalis longicornis to Babesia microti Infection and Its Ligand Identification.

Haemaphysalis longicornis is a blood-feeding hard tick known for transmitting a variety of pathogens, including Babesia How the parasites in the imbibed blood become anchored in the midgut of ticks is still unknown. Leucine-rich repeat domain (LRR)-containing protein, which is associated with the innate immune reaction and conserved in many species, has been detected in H. longicornis and has previously been indicated in inhibiting the growth of Babesia gibsoni However, the detailed mechanism is unknown. In this study, one of the ligands for LRR from H. longicornis (HlLRR) was identified in Babesia microti, designated BmActin, using glutathione transferase (GST) pulldown experiments and immunofluorescence assays. Moreover, RNA interference of HlLRR led to a decrease in the BmActin mRNA expression in the midgut of fully engorged ticks which fed on B. microti-infected mice. We also found that the expression level of the innate immune molecules in H. longicornis, defensin, antimicrobial peptides (AMPs), and lysozyme, were downregulated after the knockdown of HlLRR. However, subolesin expression was upregulated. These results indicate that HlLRR not only recognizes BmActin but may also modulate innate immunity in ticks to influence Babesia growth, which will further benefit the development of anti-Babesia vaccines or drugs.

Babesia microti Protein BmSP44 Is a Novel Protective Antigen in a Mouse Model of Babesiosis.

Babesiosis caused by Babesia species imposes an increasing threat to public-health and so far, there is no effective vaccine to prevent Babesia infections. Babesia surface antigen may participate in the invasion of erythrocytes. In our previous study, a surface antigen of B. microti merozoites, named as BmSP44 was identified as a dominant reactive antigen by protein microarray screening. To evaluate its potential applications in diagnosis and prevention of Babesiosis, the open reading frame encoding BmSP44 was cloned and the recombinant protein was expressed. In consistent with the protein microarray result, recombinant BmSP44 (rBmSP44) can be recognized by sera from B. microti infected mice. Immunofluorescence assays (IFA) confirmed that BmSP44 is a secreted protein and localized principally in the cytoplasm of the parasites. The parasitemia and Babesia gene copies were lower in mice administered rBmSP44 antisera compared with normal controls. Active immunization with rBmSP44 also afforded protection against B. microti infection. The concentrations of hemoglobin in rBmSP44 immunization group were higher than those in the control group. Importantly, vaccination of mice with rBmSP44 resulted in a Th1/Th2 mixed immune response with significantly elevated IL-10 and IFN-γ levels during the early stage of infection. Taken together, our results indicated that rBmSP44 can induce a protective immune response against Babesia infection. Thus, BmSP44 can be used as both a diagnosis marker and a vaccine candidate.

Prevalence of Infection and Co-Infection and Presence of Rickettsial Endosymbionts in Ixodes Scapularis (Acari: Ixodidae) in Connecticut, USA.

Ixodes scapularis is currently known to transmit 7 pathogens responsible for Lyme disease, anaplasmosis, babesiosis, tick-borne relapsing fever, ehrlichiosis, and Powassan encephalitis. Ixodes scapularis can also be colonized by endosymbiotic bacteria including those in the genus of Rickettsia. We screened 459 I. scapularis ticks submitted to the Connecticut Agricultural Experiment Station Tick Testing Laboratory with the objectives to (1) examine differences in infection prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia microti, and Borrelia miyamotoi, (2) evaluate whether prevalence of co-infections occur at the same frequency that would be expected based on single infection, and (3) determine the presence of rickettsial endosymbionts in I. scapularis. The prevalence of infection in I. scapularis was highest with Bo. burgdorferi sensu lato (nymph = 45.8%; female = 47.0%), followed by A. phagocytophilum (nymph = 4.0%; female = 6.9%), Ba. microti (nymph = 5.7%; female = 4.7%), and Bo. miyamotoi (nymph = 0%; female = 7.3%). We also identified rickettsial endosymbionts in 93.3% of I. scapularis. Nymphs were significantly more likely to be infected with Bo. burgdorferi if they were infected with Ba. microti, whereas adult females were significantly more likely to be infected with Bo. burgdorferi if they were infected with A. phagocytophilum. Our study suggests that the infection prevalence of Bo. burgdorferi is not independent of other co-circulating pathogens and that there is a substantially higher infection of Bo. miyamotoi in I. scapularis females compared with nymphs in this study. High prevalence of infection and co-infection with multiple pathogens in I. scapularis highlights the public health consequences in Connecticut, a state endemic for Lyme and other tick-borne diseases.

Bartonella infections in three species of Microtus: prevalence and genetic diversity, vertical transmission and the effect of concurrent Babesia microti infection on its success.

BACKGROUND: Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival. RESULTS: We sampled 124 Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos (47.1%; 40/85) and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%). CONCLUSIONS: High prevalence of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.

BmGPAC, an Antigen Capture Assay for Detection of Active Babesia microti Infection.

Human babesiosis is an emerging zoonotic infectious disease caused by intraerythrocytic protozoan parasites of the genus Babesia Most cases of human babesiosis are caused by Babesia microti and often manifest in individuals over the age of 50 years or in patients with a compromised immune system. Patients who develop symptomatic B. microti infections usually experience months of asymptomatic infection after the acute infection has resolved. About one-fifth of B. microti-infected adults never develop symptoms. These asymptomatically infected individuals sometimes donate blood and thus can transmit B. microti through blood transfusion. Current assays for detection of active B. microti infections can be used to screen donor blood prior to transfusion, but they rely primarily on microscopy or PCR methods, which have sensitivity and technical limitations. Here we report the development of an antigen capture enzyme-linked immunosorbent assay (BmGPAC) based on a major secreted immunodominant antigen of B. microti (BmGPI12/BmSA1), and we provide evidence that this assay is superior for detection of active B. microti infections, compared to available microscopy methods and serological assays. The assay has been evaluated using supernatants of B. microti-infected erythrocytes cultured in vitro, sera from B. microti-infected laboratory mice, and sera from wild mice and human patients. Our data suggest that the BmGPAC assay is a reliable assay for detection of active B. microti infections and is superior to real-time PCR and antibody assays for diagnosis of acute B. microti infections, screening of the blood supply, and epidemiological surveys of humans and animal reservoir hosts.

Screening for biomarkers reflecting the progression of Babesia microti infection.

BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.

Transplacental transmission of tick-borne Babesia microti in its natural host Peromyscus leucopus.

BACKGROUND: Babesia microti is an emerging tick-borne pathogen and the causative agent of human babesiosis. Mathematical modeling of the reproductive rate of B. microti indicates that it cannot persist in nature by horizontal tick-host transmission alone. We hypothesized that transplacental transmission in the reservoir population contributes to B. microti persistence and emergence in North American rodent populations. METHODS: Peromyscus leucopus were collected from Connecticut and Block Island, Rhode Island and analyzed using a highly specific quantitative PCR (qPCR) assay for infection with B. microti. RESULTS: In April, 100% (n = 103) of mice were infected with B. microti. Females exhibited significantly higher parasitemia than their offspring (P < 0.0001) and transplacental transmission was observed in 74.2% of embryos (n = 89). Transplacental transmission of B. microti is thus a viable and potentially important infectious pathway in naturally infected rodent species and should be considered in future theoretical and empirical studies. CONCLUSIONS: To our knowledge, this study is the first to report transplacental transmission of B. microti occurring in its natural reservoir host, P. leucopus, in the United States and the only study that provides a quantitative estimate of parasitemia. This vector-independent pathway could contribute to the increased geographic range of B. microti or increase its abundance in endemic areas.

Babesia microti-like piroplasm (syn. Babesia vulpes) infection in red foxes (Vulpes vulpes) in NW Spain (Galicia) and its relationship with Ixodes hexagonus.

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm.

Babesia microti thioredoxin 3 is an effective antioxidant and involved in the response to antiprotozoal drugs.

The intra-erythrocytic apicomplexan Babesia microti is the predominant pathogen that causes human babesiosis, an infectious disease that occurs worldwide. B. microti relies on the antioxidant including thioredoxin system to maintain the redox balance during the erythrocytic stage. In the present study, the full-length B. microti thioredoxin 3 (BmTrx3) gene was cloned, expressed in vitro, and its response to antiprotozoal drugs were tested. The full-length BmTrx3 was 663 bp and contained an intact open reading frame of 567 bp. The encoded polypeptide was 188 amino acids and the predicted molecular weight of the protein was 21.7 kDa. A conserved thioredoxin-like family domain was found in BmTrx3. The expression of BmTrx3 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. Western blot analysis showed that mouse anti-BmTrx3 serum could recognize the native BmTrx3 in parasite lysates and that the mouse anti-B. microti serum could recognize the recombinant BmTrx3 protein. Immunofluorescence microscopy showed that BmTrx3 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the enzyme activity of the purified recombinant BmTrx3 protein. The anti-malaria drug chloroquine significantly inhibited the expression of BmTrx3, however, another anti-malaria drug qunine, and a known anti-babesiosis drug clindamycin, induced significantly higher upregulation of BmTrx3 mRNA. The results of the present study demonstrate that BmTrx3 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.

Screening of patient blood samples for babesiosis using enzymatic assays.

Human babesiosis is an emerging tick-borne disease in the United States and Europe. Transmitted by Ixodes ticks, the causative agent Babesia microti is an intraerythrocytic parasite that causes mild to deadly disease. Transmission of B. microti can also occur by transfusion of infected blood and blood products resulting in transfusion-transmitted babesiosis (TTB), which carries a high risk of fatality. To effectively manage this rise in B. microti infections, better screening tools are needed, which require minimal manipulation of the samples before testing. To this end, we tested two enzymatic assays, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), for efficacy in diagnosis of babesiosis. The results show that AST and ALT activity is significantly higher in the plasma of B. microti-infected patients. Moreover, statistical analysis revealed that these assays have high sensitivity and positive predictive values, which highlights their usefulness as diagnostics for babesiosis. These standardized enzymatic assays can be used to perform high-throughput, large-scale screens of blood and blood products before they are certified safe for transfusion.

Efficacy, safety and tolerance of imidocarb dipropionate versus atovaquone or buparvaquone plus azithromycin used to treat sick dogs naturally infected with the Babesia microti-like piroplasm.

BACKGROUND: Piroplasmosis caused by the Babesia microti-like piroplasm (Bml) is increasingly being detected in dogs in Europe. Sick dogs show acute disease with severe anaemia associated with thrombocytopenia with a poor response to current available drugs. This study assesses the safety and tolerance of three treatments and compares their efficacy over a full year of follow up in dogs naturally infected with Bml. METHODS: Fifty-nine dogs naturally infected with Bml were randomly assigned to a treatment group: imidocarb dipropionate (5 mg/kg SC, 2 doses 14 d apart) (IMI); atovaquone (13.3 mg/kg PO q 8 h, 10 d)/azithromycin (10 mg/kg PO q 24 h, 10 d) (ATO); or buparvaquone (5 mg/kg IM, 2 d apart)/azithromycin (same dosage) (BUP). Before and after treatment (days 15, 45, 90 and 360), all dogs underwent a physical exam, blood tests and parasite detection (blood cytology and PCR). Clinical efficacy was assessed by grading 24 clinical and 8 clinicopathological signs from low to high severity. RESULTS: Before treatment, most dogs had severe regenerative anaemia (88.13%) and thrombocytopenia (71.4%). On treatment Day 45, clinical signs were mostly reduced in all dogs, and by Day 90, practically all dogs under the ATO or BUP regimen were clinically healthy (76.4 and 88%, respectively). Highest percentage reductions in laboratory abnormalities (82.04%) were detected in animals treated with ATO. Over the year, clinical relapse of Bml was observed in 8 dogs (8/17) treated with IMI. However, on Day 360, these animals had recovered clinically, though clinicopathological abnormalities were still present in some of them. Parasitaemia was PCR-confirmed on Days 90 and 360 in 47.05 and 50% of dogs treated with ATO, 68 and 60.08% with BUP, and 94.1 and 73.3% with IMI, respectively. Even after 360 days, 13.3% of the dogs treated with IMI returned a positive blood cytology result. CONCLUSIONS: IMI showed the worse clinical and parasitological, efficacy such that its use to treat Bml infection in dogs is not recommended. The treatments ATO and BUP showed better efficacy, though they were still incapable to completely eliminate PCR-proven infection at the recommended dose. All three treatments showed good tolerance and safety with scarce adverse events observed.

Identification of a thioredoxin reductase from Babesia microti during mammalian infection.

Babesia microti is the primary causative agent of human babesiosis worldwide and associated with increased human health risks and the safety of blood supply. The parasite replicates in the host's red blood cells, thus, in order to counteract the oxidative stress and toxic effects, parasites employ a thioredoxin (Trx) system to maintain a redox balance. Since thioredoxin reductase (TrxR) plays a critical role in the system, in this study, we report the cloning, expression, and functional characterization of a novel TrxR from B. microti (BmiTrxR). The complete gene BmiTrxR was obtained by amplifying the 5' and 3' regions of messenger RNA (mRNA) by RACE. The full-length complementary DNA (cDNA) of BmiTrxR was 1766 bp and contained an intact open reading frame with 1662 bp that encoded a polypeptide with 553 amino acids. Molecular weight of the predicted protein was 58.4 kDa with an isoelectric point of 6.95, similar to high molecular weight TrxR. The recombinant protein of BmiTrxR was expressed in a His-fused soluble form in Escherichia coli. The native protein BmiTrxR was identified with the mouse anti-BmiTrxR polyclonal serum by western blotting and IFAT. Moreover, the enzyme showed a disulfide reductase activity using DTNB as substrate and catalyzed the NADPH-dependent reduction of Trx. Auranofin, a known inhibitor of TrxR, completely abrogated the activity of the recombinant enzyme in vitro. These results not only contribute to the understanding of redox pathway in this parasite but also suggest that BmiTrxR could be a potential target for the development of novel strategies to control B. microti thus reducing the incidence of babesiosis.

Experimental transmission of Babesia microti by Rhipicephalus haemaphysaloides.

BACKGROUND: Human babesiosis is considered an emerging threat in China. Dozens of human infections with Babesia microti have been reported recently, especially in southern China. However, the transmission vectors of this parasite in these areas are not well understood. Rhipicephalus haemaphysaloides, which is one of the dominant tick species in southern China, is a major vector of bovine babesiosis in China. However, whether this tick has the potential to transmit B. microti has not been tested. The present study experimentally investigated the transmission competence of B. microti through R. haemaphysaloides ticks. METHODS: Larvae and nymphs of R. haemaphysaloides ticks were fed on laboratory mice infected by B. microti. The infection was detected by PCR at 4 weeks post-molting. BALB/c and NOD/SCID mice were infested by nymphs molting from larvae that ingested the blood of infective mice, and blood samples were then analyzed by PCR. RESULTS: Experimental transstadial transmission of R. haemaphysaloides for B. microti was proved in both the larvae to nymph and the nymph to adult transstadial routes. The positive rate of B. microti was 43.8% in nymphs developed from larvae consumed infected mice and 96.7% in adults developed from nymphs exposed to positive mice. Among the mice infested by infective nymphs, B. microti was detected in 16.7% (2/12) of the BALB/c mice and in all of the NOD/SCID (6/6). However, the parasite was not observed to persist beyond more than one molt, and transovarial transmission did not occur. CONCLUSIONS: This is the first study to demonstrate that B. microti can be transmitted artificially by R. haemaphysaloides. This tick species might be a potential vector of human babesiosis in southern China, which represents a public health concern.

Analysis of main T-cell subsets and activated T supressor/cytotoxic cells in patients with Borrelia burgdorferi s. lato only infection and co-infections with Anaplasma phagocytophilum, Bartonella spp. and Babesia microti.

INTRODUCTION: The study was designed to assess the role of some important immunologic factors with regards to both laboratory results and clinical symptoms in patients with confirmed Lyme disease. Additional examinations were carried out for co-infections with a number of tick-borne pathogens. MATERIAL AND METHODS: The study group consisted of 54 patients with Lyme disease and a group of 21 healthy controls. Serology of co-infections with Anaplasma phagocytophilum, Bartonella spp. and Babesia microti was carrieed out in all patients. Blood samples were stained using the whole-blood lysis method and analyzed concurrently on a flow cytometer FACSCalibur. Directly conjugated anti-human monoclonal antibodies against CD3, CD4, CD8, CD16, CD56, HLA-DR and CD69 were used. RESULTS: No significant differences were observed with respect to thepretreatment level of CD4+ and CD8+ cells. In patients with symptoms relief and symptoms persistence, lower percentages of CD4+ and CD8+ cells were found, but with no statistical dependence. In the study group, both in patients with and without co-infections, pretreatment values of CD16+CD56+ cells did not differ significantly. In patients who did not respond to the treatment, the baseline percentage of NK cells was higher (P<0.01) than in group with clinical improvement, and lower after the treatment, whereas in patients with symptoms relief after the treatment there was an increase in the percentage of NK cells. CONCLUSION: Co-infections with Anaplasma phagocytophilum, Bartonella spp. and Babesia microti had no impact on T-cell percentages in Lyme disease patients. There was a lower baseline percentage of NK cells in patients not responding to antibiotic treatment.

Coinfection by Ixodes Tick-Borne Pathogens: Ecological, Epidemiological, and Clinical Consequences.

Ixodes ticks maintain a large and diverse array of human pathogens in the enzootic cycle, including Borrelia burgdorferi and Babesia microti. Despite the poor ecological fitness of B. microti, babesiosis has recently emerged in areas endemic for Lyme disease. Studies in ticks, reservoir hosts, and humans indicate that coinfection with B. burgdorferi and B. microti is common, promotes transmission and emergence of B. microti in the enzootic cycle, and causes greater disease severity and duration in humans. These interdisciplinary studies may serve as a paradigm for the study of other vector-borne coinfections. Identifying ecological drivers of pathogen emergence and host factors that fuel disease severity in coinfected individuals will help guide the design of effective preventative and therapeutic strategies.

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report.

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1st day of Babesia infection (Group G0); on the 28th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90th and 150th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01-0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.