Search Results (137)

Background: Bovine babesiosis, caused by the tick-borne apicomplexan parasite Babesia spp., is an economically significant disease that threatens the cattle industry worldwide. Babesia bovis is the most pathogenic species, leading to high morbidity and mortality in infected animals. One promising approach to vaccination against bovine babesiosis involves the use of multiple protective antigens, offering advantages over traditional live-attenuated vaccines. Tools such as immunobioinformatics and reverse vaccinology have facilitated the identification of novel antigens. Enolase, a “moonlighting” enzyme of the glycolytic pathway with demonstrated vaccine potential in other pathogens, has not yet been studied in B. bovis. Methods: In this study, the enolase gene from two B. bovis isolates was successfully identified and sequenced. The gene, consisting of 1366 base pairs, encodes a predicted protein of 438 amino acids. Its expression in intraerythrocytic parasites was confirmed by RT-PCR. Two peptides containing predicted B-cell epitopes were synthesized and used to immunize rabbits. Hyperimmune sera were then analyzed by ELISA, confocal microscopy, Western blot, and an in vitro neutralization assay. Results: The hyperimmune sera showed high antibody titers, reaching up to 1:256,000. Specific antibodies recognized intraerythrocytic merozoites by confocal microscopy and bound to a ~47 kDa protein in erythrocytic cultures of B. bovis as detected by Western blot. In the neutralization assay, antibodies raised against peptide 1 had no observable effect, whereas those targeting peptide 2 significantly reduced parasitemia by 71.99%. Conclusions: These results suggest that B. bovis enolase contains B-cell epitopes capable of inducing neutralizing antibodies and may play a role in parasite–host interactions. Enolase is therefore a promising candidate for further exploration as a vaccine antigen. Nonetheless, additional experimental studies are needed to fully elucidate its biological function and validate its vaccine potential. Full article

Rhipicephalus microplus is an important biological vector as it transmits several pathogens, including Babesia bovis, the causative agent of bovine babesiosis. The available strategies for controlling B. bovis are limited, resulting in substantial challenges for both animal health and livestock management. Infection of the tick midgut is the essential first step for the transmission cycle of B. bovis, yet this process remains largely unexamined. To better understand the first step of tick infection, this study employed a proteomic approach to identify a midgut protein that responds to B. bovis infection. We then used RNA interference for gene silencing to determine if the protein is essential for R. microplus infection. The protein we identified, Rm24, is twofold upregulated in the tick midgut during B. bovis infection. We silenced the gene encoding Rm24 and examined the effect of reduced expression on both tick fitness and B. bovis infection. Our results indicated that silencing the Rm24 gene impacted the survivability of adult female ticks, which exhibited a significant reduction in viability as compared to the control and non-injected groups. Importantly, we found that suppressing the gene encoding Rm24 led to a significant decrease in the number of engorged female ticks infected, with only 15% of female ticks testing positive for B. bovis kinetes as compared to over 50% in the control groups. We also detected a significant reduction in vertical transmission of B. bovis to larval progenies. These findings suggest that the Rm24 protein is critical for infection by B. bovis and could serve as a promising target for future transmission-blocking strategies. Full article

In order to investigate the infection status of Cryptosporidium in O. curzoniae and B. grunniens in Zoige County, Sichuan Province, fecal samples from B. grunniens and gastrointestinal contents from captured O. curzoniae were collected between March and December 2023 from five townships (Dazhasi, Axi, Hongxing, Tangke, and Maixi). Genomic DNA was extracted, and nested PCR targeting the small subunit (SSU) rRNA gene of Cryptosporidium was performed. PCR-positive products were sequenced, trimmed, aligned, and subjected to phylogenetic analysis to determine species and genotypes. A total of 242 samples were obtained, of which 20 were Cryptosporidium SSU rRNA-positive, yielding an overall detection rate of 8.3% (20/242). The detection rates of O. curzoniae and B. grunniens were 7.0% (8/114) and 9.4% (12/128), respectively. Among the five sampling sites, Maixi town exhibited the highest detection rate (32.4%, 11/44), followed by Hongxing town (15.2%, 7/46) and Tangke town (4.6%, 2/44). Phylogenetic analysis detected an unidentified Cryptosporidium sp. in O. curzoniae, while C. bovis (n = 10) and C. ryanae (n = 2) were detected in B. grunniens. These findings demonstrate that Cryptosporidium infections are present in both O. curzoniae and B. grunniens in Zoige County, with notable differences in infection rates and species composition. Continued surveillance of Cryptosporidium in local livestock and wildlife is warranted to provide critical data for regional public health management. Full article

Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella multocida (PM), Mannheimia haemolytica (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes. Full article

Mycobacterium bovis is the causative pathogen of bovine tuberculosis (bTB), a disease that affects cattle and other mammals, including humans. Currently, there is no efficient vaccine against bTB, underscoring the need for novel immunization strategies. The M72 fusion protein, composed of three polypeptides derived from Mycobacterium tuberculosis and M. bovis, has demonstrated protective efficacy against M. tuberculosis in clinical trials when combined with the AS01E adjuvant. Given the established efficacy of nanocapsule formulations as vaccine delivery systems, this study evaluated a novel immunization strategy combining BCG with either full-length M72 or a truncated M72 fused to a streptococcal albumin-binding domain (ABDsM72). Both antigens were encapsulated in chitosan/alginate nanocapsules and assessed in a murine M. bovis challenge model. Priming with BCG followed by an M72 boost significantly improved splenic protection compared to BCG alone, but it did not enhance pulmonary protection. Notably, boosting with ABDsM72 further increased the proportion of CD4+KLRG1-CXCR3+ T cells in the lungs of M. bovis-challenged mice, a key correlate of protective immunity. These findings demonstrate that chitosan/alginate-encapsulated antigens enhance BCG-induced immunity, supporting their potential as next-generation vaccine candidates for bTB control. Full article

Mycobacterium tuberculosis (Mtb) is a major global health threat, exacerbated by the emergence of antibiotic-resistant strains. This study investigated fluoroquinolone resistance protein A (MfpA), which enhances mycobacterial survival by targeting host mitochondria and regulating apoptosis. Wild-type (WT) and knockout (KO) Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strains, a common model for Mtb, were utilized to examine host cell responses. Compared to WT strains, KO strains showed reduced colony-forming units (CFUs), elevated TNF-α and IL-6 levels, and increased apoptosis. MfpA was found to localize to mitochondria, increasing ROS production and disrupting mitochondrial membrane potential. Transcriptomic analysis revealed that MfpA modulated the NF-κB signaling pathway, regulating the expression of gadd45β. These results suggest that MfpA drives both antibiotic resistance and virulence by suppressing apoptosis via the mitochondrial and NF-κB pathways, promoting mycobacterial persistence. Studies using BCG provide valuable insight into Mtb’s survival mechanisms, highlighting MfpA’s dual role in resistance and pathogenesis. Full article

Background: The prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing in Europe and North America. Most pulmonary NTM cases are caused by Mycobacterium avium complex (MAC). The treatment of pulmonary MAC is suboptimal with failure rates ranging from 30% to 40% and there is a need to develop new vaccines. Methods: We tested the ability of two whole-cell vaccines, DAR-901 (heat-killed M. obuense) and BCG (live-attenuated M. bovis), to induce MAC cross-reactive immunity by first immunizing BALB/c mice and then performing IFN-γ ELISPOT assays after overnight stimulation of splenocytes with live MAC. To study the ability of these vaccines to protect against MAC infection, BALB/c mice were vaccinated with DAR-901 (intradermal) or BCG (subcutaneous or intranasal) and challenged with aerosolized MAC 4 weeks later. A group of mice vaccinated with BCG were also treated with clarithromycin via gavage. Lung colony-forming units (CFU) in immunized mice and unvaccinated controls were quantified 4 weeks after infection. Histopathology was used to quantify lung inflammation and flow cytometry was used to study lung immunity in BCG-vaccinated and unvaccinated mice following MAC infection. To increase the safety profile of mucosal BCG vaccination, we studied BCG with a “kill switch” (tetR BCG) in scnn1b-transgenic mice (i.e., mice prone to cystic fibrosis-type lung diseases). Results: Our results showed that (i) DAR-901 induced cross-reactive immunity to MAC to a similar level as BCG, (ii) DAR-901 and BCG protected against aerosol MAC challenge, (iii) mucosal BCG vaccination, compared to systemic BCG and DAR-901 vaccinations, provided the best protection against MAC challenge, (iv) BCG vaccination did not interfere with anti-MAC activities of clarithromycin, (v) BCG-vaccinated mice had increased inflammation and increased frequencies of activated CD4 and CD8 T cells following MAC infection, and (vi) doxycycline treatment of tetR BCG-vaccinated mice decreased lung BCG CFU without affecting MAC immunity. Conclusions: Both DAR-901 and BCG vaccinations induce MAC cross-reactive immunity and protect against aerosolized MAC challenges. Mucosal BCG vaccination provides the best protection and TetR BCG could enhance the safety of mucosal BCG vaccination. Full article

Mastitis is a common condition in dairy cattle that causes huge losses globally. The inflammation is caused by the invasion of the teat canal by pathogens, including hard-to-control single-cell microalgae of the genus Prototheca. The aim of the study was the in vitro comparison of the antimicrobial properties of 10 selected essential oils (EOs) and amphotericin B (AMB) against Prototheca bovis strains (PRO3 and PRO7) from different regions in Poland. The antialgal effect was estimated by using toxicity tests. The chemical composition of the EOs was determined by using gas chromatography coupled with mass spectrometry. The tested EOs had significant cytotoxic effects on algal viability. A statistical analysis of the results revealed that the highest biocidal potential, at a concentration of 2%, was demonstrated by lavender, rosemary, and oregano oils, reducing the survival of the Prototheca bovis strains, on average, by 51.21%, 45.83%, and 45.15%, respectively. In comparison, AMB reduced algal viability by an average of 88% compared with the control groups. Further research into the utilization of the biocidal properties of lavender, rosemary, and oregano oil against Prototheca spp. may help to develop new forms of treatments against mastitis caused by this pathogen in the future. Full article

Background: Bovine tuberculosis (bTB) is an infectious disease which causes significant damage to the farming industry and remains a disease of global significance. Although control strategies have focused on a test and cull approach primarily based around specific cell-mediated immune responses, serological assays are increasingly being used as a supplementary test alongside skin testing and interferon-gamma release (IGRA) assays. The UK is moving towards the use of the Bacillus Calmette–Guérin (BCG) vaccination of cattle as an additional targeted control tool against bTB. However, there are concerns over its potential impact on the outcomes of bTB diagnostic tests and other non-TB assays, such as serological tests for Mycobacterium avium subsp. paratuberculosis (MAP). Methods: We investigated the performance of commercially available serology tests designed to detect bTB and MAP using serum samples from BCG-vaccinated animals which were subsequently infected with Mycobacterium bovis (M. bovis). Results: BCG vaccination per se did not significantly impact the specificity of serological diagnostic tests for bTB or Johne’s disease. However, increased numbers of false-positive responses in bTB serology tests were seen in BCG-vaccinated animals 3 weeks following a tuberculin skin test, where up to 23% and 54% of animals gave a positive result in IDEXX and Enferplex tests, respectively. Furthermore, M. bovis infection gave rise to false-positive test results for Johne’s disease, irrespective of the animals’ prior BCG vaccination status. Conclusions: Caution should be taken when assessing results from serology tests for bTB if tuberculin skin testing has occurred shortly before collection of blood from BCG-vaccinated cattle. Furthermore, these results highlight the potential for misdiagnosis of MAP infection when using serology tests in bTB-infected cattle. Full article

Ticks are major vectors of numerous pathogens, and their public health significance is increasing due to climate change and the expanding involvement of wildlife hosts. In Korea, the Korean water deer (KWD, Hydropotes inermis argyropus) is widely distributed and considered a key wild host for ticks, potentially serving as a reservoir for diverse tick-borne diseases (TBDs). To examine the geographical and temporal distribution of TBDs in this species, 1035 spleen samples were collected from KWDs across 12 regions between April and November 2023. Overall, 93.6% (969) of samples were positive for at least one pathogen. Five Anaplasma species were identified: A. phagocytophilum, A. phagocytophilum-like A (APLA), APLB, A. bovis, and A. capra. In addition, Borrelia theileri and three Theileria species—T. capreoli, T. cervi, and T. luwenshuni—were detected. Notably, this study presents the first global detection of APLA, APLB, and B. theileri in KWDs. Statistically significant regional and seasonal differences in prevalence were observed, with higher detection rates in northern regions and during autumn, suggesting that environmental and ecological factors influence transmission dynamics. These findings highlight the role of KWD as a reservoir host for multiple TBDs and underscore the importance of wildlife-based surveillance for assessing zoonotic disease risks. Full article

Babesia bovis is a protozoan parasite that causes babesiosis in cattle. It has been hypothesized that in apicomplexan parasites, translationally controlled tumor protein (TCTP) interferes with the host immune response by inhibiting B cell proliferation. The aim of this study was the characterization of B. bovis TCTP (BboTCTP) and the evaluation of its expression, immunogenicity and role in infection. The tctp gene was identified and sequenced from B. bovis isolates and revealed a high conservation. Expression was confirmed in intraerythrocytic stages by Western blot and confocal microscopy. Synthetic peptides containing predicted B cell epitopes were used to immunize cattle, followed by a challenge with a virulent B. bovis strain. Immunized animals showed milder clinical signs and faster recovery compared to controls. Sera from non-immunized animals exhibited lower total IgG levels after challenge (p < 0.05), while sera from immunized animals induced significant in vitro invasion inhibition (32–33%). These results suggest that BboTCTP is immunogenic and may play a role in modulating the host immune response. The results provide novel insights into B. bovis biology and support BboTCTP as a promising candidate for further evaluation as a vaccine antigen. Future studies should explore its immunomodulatory mechanisms and potential use in combined vaccine formulations. Full article

This study evaluated the effects of various concentrations of PVP-40 on the in vitro cryopreservation and recovery of Babesia bovis and Babesia bigemina. We also assessed a reduced dose of attenuated Babesia strains to determine its efficacy in preventing clinical disease. A microaerophilic stationary phase blood culture system was used to recover Babesia parasites that were cryopreserved in solutions with various PVP-40 concentrations and Babesia parasites in 20% PVP-40 were used to vaccinate naïve cattle. The animals were vaccinated intramuscularly with frozen parasites cryopreserved in 20% PVP-40, with a dose of either 1 × 10⁸ or 1 × 10⁷ erythrocytes infected with both attenuated B. bigemina and B. bovis produced from blood cultures. The control group received uninfected erythrocytes. During the vaccination, clinical parameters such as rectal temperature and hematocrit levels were unaffected. The animals were relocated to a farm in a Babesia hyperendemic area to test the efficacy of these live vaccines in controlling disease onset. Some vaccinated animals showed mild disease. In the vaccinated groups, parasites were detected in blood smears for only one day during the challenge. In contrast, the control group experienced fever for three consecutive days, a decline in hematocrit levels, and significant health deterioration. In this group, parasites were detected in smears for four consecutive days. All the animals in the control group required treatment to manage their high parasitemia and prevent mortality. In this study, we demonstrated that increasing the concentration of PVP-40 to cryopreserve parasites improved the recovery and proliferation of Babesia spp. in blood culture, and we also showed that when animals were vaccinated with cryopreserved, in vitro cultured, attenuated Babesia parasites in 20% PVP-40, they were effectively protected from severe clinical babesiosis. Full article

Bartonellae are zoonotic pathogens with a broad range of reservoir hosts and vectors. To examine sylvatic Bartonella reservoirs, tissue samples of red deer (Cervus elaphus, n = 114) and their associated deer keds (Lipoptena cervi, n = 50; L. fortisetosa, n = 272) collected in the Czech Republic were tested for the presence of Bartonella using PCR at four loci (gltA, rpoB, nuoG, ITS); PCR sensitivity was increased significantly by using primers modified for the detection of wildlife-associated bartonellae. One-third of the deer and 70% of the deer keds were Bartonella positive; within the tested animal tissues, usually the spleen was positive. The most prevalent Bartonella represents an undescribed species related to isolates from Japanese sika deer and L. fortisetosa. Additionally, B. schoenbuchensis sensu lato and B. bovis were found, together making up 17 genotypes characterized by multi-locus sequence typing, all unique compared to previously published sequences. Nanopore sequencing of selected samples revealed an additional 14 unique Bartonella genotypes, with up to six genotypes co-infecting one deer, highlighting the diversity of ruminant Bartonella. The high COI variety of examined L. cervi and L. fortisetosa suggests L. fortisetosa in central Europe is not a homogenous invasive population. Full article

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis), remains the leading cause of death from a single infectious agent globally. Intracellular survival is crucial for their virulence; yet, the underlying mechanisms are not fully understood. This study aimed to demonstrate the significance of a previously unannotated bovine gene ENSBTAG00000011305 in M. bovis intracellular survival. This gene was termed NMRAL2_Bovine due to its inclusion of the NmrA domain which has a relation to nitric oxide (NO) production. We used CRISPR/Cas9 to knock out NMRAL2_Bovine in bovine lung epithelial cells and observed a significant decrease in M. bovis-induced cell death and the intracellular bacterial count, alongside increased NO levels. A transcriptome analysis revealed the upregulation of pathways linked to NO, IL-6, and TNF-α production, which was confirmed by the increased expression of iNOS, IL-6, and TNF-α. Correspondingly, Western blotting indicated that key signaling pathways, including NF-κB and MAPK, were activated. In conclusion, our findings determined that NMRAL2_Bovine functions as a negative regulator of the inflammatory response induced by M. bovis infection at the cellular level and, thereby, provide a novel insight into TB pathogenesis and a potential target for developing novel host-directed therapies against TB. Full article

Background/Objectives: Bovine babesiosis is a vector-borne disease transmitted by ticks that causes important losses in livestock worldwide. Recent research performed on the drugs currently used to control bovine babesiosis reported several issues including drug resistance, toxicity impact, and residues in edible tissue, suggesting the need for developing novel effective therapies. The endochin-like quinolones ELQ-316 and buparvaquone (BPQ) act as cytochrome bc1 inhibitors and have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp. and Babesia microti, without showing toxicity in mammals. The objectives of this study are investigating whether ELQ-316, BPQ, and their combination treatment could be effective against Babesia bovis in an in vitro culture model and comparing with imidocarb (ID), the routinely used drug. Methods: In vitro cultured parasites starting at 2% percentage of parasitemia (PPE) were treated with BPQ, ELQ-316, ID, and the combinations of BPQ + ELQ-316 and ID + ELQ-316 at drug concentrations that ranged from 25 to 1200 nM, during four consecutive days. The IC50% and IC99% were reported. Parasitemia levels were evaluated daily using microscopic examination. Data were compared using the non-parametrical Mann–Whitney and Kruskall–Wallis test. Results: All drugs tested, whether used alone or in combination, significantly decreased the survival (p < 0.05) of B. bovis in in vitro cultures. The combination of BPQ + ELQ-316 had the lowest calculated inhibitory concentration 50% (IC50%) values, 31.21 nM (IC95%: 15.06–68.48); followed by BPQ, 77.06 nM (IC95%: 70.16–86.01); ID + ELQ316, 197 nM (IC95%:129.0–311.2); ID, 635.1 nM (IC95%: 280.9–2119); and ELQ316, 654.9 nM (IC95%: 362.3–1411). Conclusions: The results reinforce the higher efficacy of BPQ at affecting B. bovis survival and the potential synergistic effects of its combination with ELQ-316, providing a promising treatment option against B. bovis. Full article