Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development of ruminant animal models of infection is urgently needed to evaluate therapeutic and prophylactic tools. Herein, we ...studied the effect of parasite dose and host age on the infection dynamics with Besnoitia besnoiti tachyzoites in cattle in two independent experimental infections. In experiment A, twelve 3‐month‐old male calves were inoculated intravenously with either three different doses of tachyzoites (G1: 108; G2: 107; G3: 106) or with PBS (G4). In experiment B, six 14‐month‐old bulls were inoculated with 106 tachyzoites based on results obtained in experiment A. In both trials, clinical signs compatible with acute and chronic besnoitiosis were monitored daily; blood and skin samples were collected regularly for 70–115 days post‐infection (pi). Finally, animals were killed, and tissues were collected for lesion and parasite detections. Infected animals developed mild–moderate signs compatible with acute besnoitiosis. Lymphadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) and bulls (from 6 days until 9 days pi). Seroconversion was detected at 16–19 days pi, and antibody levels remained high. Infected animals did not developed characteristic clinical signs and macroscopic lesions of chronic besnoitiosis. However, successfully, parasite‐DNA was detected in a reduced number of target tissues: conjunctiva, ocular sclera, epididymis, skin of the scrotum and carpus in calves (n = 10, 6 of which belonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remarkably, one tissue cyst and mild microscopic lesions were also detected. In summary, inoculated animals developed the acute besnoitiosis and chronic infection was evidenced by microscopic findings. However, our results suggest that tachyzoite dose and host age are not key variables for inducing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Thus, a further refinement of this model should evaluate other parasite‐ and host‐dependent variables.
Bovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control ...tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce.
The objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs.
Morphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3-4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs.
We have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.
Neonatal diarrhoea in calves is one of the major health problems in the cattle industry. Although co-infections are often associated with greater severity of disease, there is limited information on ...any impact on the pathogens themselves. Herein, we studied Cryptosporidium parvum and bovine coronavirus (BCoV) in human HCT-8 cells, inoculated either sequentially or simultaneously, to investigate any influence from the co-infections. Quantitative results from (RT)-qPCR showed that prior inoculation with either of the two pathogens had no influence on the other. However, the results from simultaneous co-inoculation showed that entry of viral particles was higher when C. parvum sporozoites were present, although elevated virus copy numbers were no longer evident after 24 h. The attachment of BCoV to the sporozoites was probably due to specific binding, as investigations with bovine norovirus or equine herpes virus-1 showed no attachment between sporozoites and these viruses. Flow cytometry results at 72 h post inoculation revealed that C. parvum and BCoV infected 1–11% and 10–20% of the HCT-8 cells, respectively, with only 0.04% of individual cells showing double infections. The results from confocal microscopy corroborated those results, showing an increase in foci of infection from 24 to 72 h post inoculation for both pathogens, but with few double infected cells.
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•Besnoitia besnoiti can invade and proliferate in primary bovine macrophages.•Besnoitia besnoiti induced morphological and transcriptomic changes in macrophages.•Host cell enriched ...pathways and parasite effectors were identified by dual RNA-Seq analysis.•Modulation of apoptosis and Herpes simplex virus 1 infection pathways could facilitate parasite dissemination.•Numerous rhoptry protein encoding genes were upregulated at 8 h p.i.
Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.
Objectives
Morphological and molecular approaches were used to identify arthropods associated with feline pruritus. The literature associated with the arthropod genus identified was reviewed.
Methods
...On two occasions (summer 2020 and summer 2021), the owner of a cat with seasonal pruritus (commenced 2020) found the cat’s bed was substantially infested with arthropods suspected of being associated with exacerbated pruritus. The pruritus was largely itching and hair loss, particularly on the abdomen, and flaking skin patches. On the second occasion (2021), examples of the arthropods were sent to the parasitology laboratory at the Norwegian University of Life Sciences for identification. They were examined by stereomicroscopy and tentatively identified based on morphology. DNA was extracted, and identification confirmed by PCR and sequencing. The literature was reviewed to determine whether this arthropod genus has previously been considered to be associated with pruritus or the infestation of mammals.
Results
Based on morphological characteristics, the arthropods were tentatively identified as Nothrus species mites. This was confirmed by PCR. A literature review found no previous reports of pruritus or other clinical signs associated with Nothrus species mites, and mites were not found on the cat. However, this mite has previously been found on small mammals at densities exceeding that expected of incidental stragglers.
Conclusions and relevance
The large number of Nothrus species mites could have exacerbated the cat’s pruritus. By publishing this study, we hope to alert veterinarians to the possibility that Nothrus species mites may cause or exacerbate pruritus in cats.
•A panel of commercially available drugs in cattle were tested against Besnoitia besnoiti in vitro.•Decoquinate and diclazuril showed the best results in terms of efficacy.•Decoquinate primarily ...affected the parasite mitochondrium.•Diclazuril interfered in cytokinesis of daughter zoites.•Decoquinate and diclazuril treatments do not act parasiticidal.
Repurposing of currently marketed compounds with proven efficacy against apicomplexan parasites was used as an approach to define novel candidate therapeutics for bovine besnoitiosis. Besnoitia besnoiti tachyzoites grown in MARC-145 cells were exposed to different concentrations of toltrazuril, diclazuril, imidocarb, decoquinate, sulfadiazine and trimethoprim alone or in combination with sulfadiazine. Drugs were added either just prior to infection of MARC-145 cells (0 h post infection, hpi) or at 6 hpi. A primary evaluation of drug effects was done by direct immunofluorescence staining and counting. Potential effects on the host cells were assessed using a XTT kit for cell proliferation. Compounds displaying promising efficacy were selected for IC50 and IC99 determination by qPCR. In addition, the impact of drugs on the tachyzoite ultrastructure was assessed by TEM and long-term treatment assays were performed. Cytotoxicity assays confirmed that none of the compounds affected the host cells. Decoquinate and diclazuril displayed invasion inhibition rates of 90 and 83% at 0 h pi and 73 and 72% at 6 h pi, respectively. The remaining drugs showed lower efficacy and were not further studied. Decoquinate and diclazuril exhibited IC99 values of 100 nM and 29.9 μM, respectively. TEM showed that decoquinate primarily affected the parasite mitochondrium, whilst diclazuril interfered in cytokinesis of daughter zoites. The present study demonstrates the efficacy of diclazuril and decoquinate against B. besnoiti in vitro and further assessments of safety and efficacy of both drugs should be performed in the target species.
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•The Besnoitia besnoiti CDPK1 orthologue was identified and cloned.•Activity of recombinant BbCDPK1 was greatly inhibited by a panel of bumped kinase inhibitors (BKIs).•BKIs 1294, ...1517, 1553 and 1571 inhibited tachyzoite invasion and proliferation with a parasitostatic effect.•BKIs may be effective candidates to control B. besnoiti infection.
Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.
•Wild ruminant sera (n=2608) from areas where bovine besnoitiosis is present were tested.•Only one red deer and one roe deer were proved to be seropositive.•Besnoitia spp. DNA was detected in a lymph ...node from a seropositive red deer.•Microsatellite analysis suggested B. besnoiti identity.•Wild ruminants do not pose a risk of transmitting the infection to cattle.
Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n=309), roe deer (n=417), Pyrenean chamois (Rupicapra p. pyrenaica, n=383) and Iberian wild goat (Capra pyrenaica hispanica, n=288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n=820), roe deer (n=37), fallow deer (Dama dama, n=166), Iberian wild goat (n=86) and European mouflon (Ovis orientalis musimon, n=102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.
Endothelial injury, inflammatory infiltrate and fibrosis are the predominant lesions in the testis of bulls with besnoitiosis that may result in sterility. Moreover, fibroblasts, which are key ...players in fibrosis, are parasite target cells in a Besnoitia besnoiti chronic infection. This study aimed to decipher the molecular basis that underlies a drift toward fibrosis during the disease progression. Transcriptomic analysis was developed at two times post-infection (p.i.), representative of invasion (12 h p.i.) and intracellular proliferation (32 h p.i.), in primary bovine aorta fibroblasts infected with B. besnoiti tachyzoites. Once the enriched host pathways were identified, we studied the expression of selected differentially expressed genes (DEGs) in the scrotal skin of sterile infected bulls. Functional enrichment analyses of DEGs revealed shared hallmarks of cancer and early fibrosis. Biomarkers of inflammation, angiogenesis, cancer, and MAPK signaling stood out at 12 h p.i. At 32 h p.i., again MAPK and cancer pathways were enriched together with the PI3K–AKT pathway related to cell proliferation. Some DEGs were also regulated in the skin samples of naturally infected bulls (PLAUR, TGFβ1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.
Antecedentes: la albahaca (Ocium basilicum) es una hierba perteneciente a la familia laminaceae, caracterizada por sus bondades medicinales. Se ha referido su uso en la terapéutica del cáncer, ...vitíiigo, hipercolesterolemia y la diabetes mellitus.
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